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Sample records for affinity fluorescent znii

  1. Synthesis, fluorescence study and biological evaluation of three Zn(II) complexes with Paeonol Schiff base.

    PubMed

    Qin, Dong-dong; Yang, Zheng-yin; Qi, Gao-fei

    2009-10-01

    The synthesis of three Paeonol Schiff base ligand and their Zn(II) complexes are reported. The complexes were fully characterized by IR, (1)H NMR, elemental analysis and molar conductivity. The experiment results show the three Zn(II) complexes can emit bright fluorescence at room temperature in DMF solution and solid state. The fluorescence quantum yields (Phi) of three Schiff base ligands and their Zn(II) complexes were calculated using quinine sulfate as the reference with a known Phi(R) of 0.546 in 1.0N sulfuric acid. Furthermore, in order to develop these Zn(II) complexes' biological value, the antioxidant activities against hydroxyl radicals (OH*) were evaluated. The results show the three complexes possess excellent ability to scavenge hydroxyl radicals. PMID:19632146

  2. Synthesis, fluorescence study and biological evaluation of three Zn(II) complexes with Paeonol Schiff base

    NASA Astrophysics Data System (ADS)

    Qin, Dong-dong; Yang, Zheng-yin; Qi, Gao-fei

    2009-10-01

    The synthesis of three Paeonol Schiff base ligand and their Zn(II) complexes are reported. The complexes were fully characterized by IR, 1H NMR, elemental analysis and molar conductivity. The experiment results show the three Zn(II) complexes can emit bright fluorescence at room temperature in DMF solution and solid state. The fluorescence quantum yields ( Φ) of three Schiff base ligands and their Zn(II) complexes were calculated using quinine sulfate as the reference with a known ΦR of 0.546 in 1.0N sulfuric acid. Furthermore, in order to develop these Zn(II) complexes' biological value, the antioxidant activities against hydroxyl radicals (OH rad ) were evaluated. The results show the three complexes possess excellent ability to scavenge hydroxyl radicals.

  3. A target-induced fluorescent nanoparticle for in situ monitoring of Zn(II).

    PubMed

    John, Carrie L; Huan, Yanfu; Wu, Xu; Jin, Yuhui; Pierce, David T; Zhao, Julia Xiaojun

    2013-09-01

    A target-induced fluorescent silica nanoparticle has been developed for the identification, enrichment and in situ determination of trace amounts of zinc(II). The nanoparticle combines the advantages of target-induced fluorescent compounds and the small size of the nanomaterial to produce a new, smarter nanosignaling material that is capable of selectively enriching a target and detecting a specific binding process in one step. As the target analyte, Zn(II), changes the fluorescence characteristics of the nanoparticle and effectively 'turns on' the fluorescence signal, no separation step is needed to confirm or quantify the binding process. The designed nanoparticle was characterized by several aspects prior to monitoring of Zn(II) in situ. The interferences from common metal ions were studied in detail. The photostability and reversibility of the sensing materials were investigated as well. The ability of this nanoparticle to detect the target Zn(II) provides a great advantage for in situ monitoring targets in biological samples under the fluorescence microscope. PMID:23799230

  4. A fluorescent chemosensor for Zn(II). Exciplex formation in solution and the solid state.

    PubMed

    Bencini, Andrea; Berni, Emanuela; Bianchi, Antonio; Fornasari, Patrizia; Giorgi, Claudia; Lima, Joao C; Lodeiro, Carlos; Melo, Maria J; de Melo, J Seixas; Parola, Antonio Jorge; Pina, Fernando; Pina, Joao; Valtancoli, Barbara

    2004-07-21

    The macrocyclic phenanthrolinophane 2,9-[2,5,8-triaza-5-(N-anthracene-9-methylamino)ethyl]-[9]-1,10-phenanthrolinophane (L) bearing a pendant arm containing a coordinating amine and an anthracene group forms stable complexes with Zn(II), Cd(II) and Hg(II) in solution. Stability constants of these complexes were determined in 0.10 mol dm(-3) NMe(4)Cl H(2)O-MeCN (1:1, v/v) solution at 298.1 +/- 0.1 K by means of potentiometric (pH metric) titration. The fluorescence emission properties of these complexes were studied in this solvent. For the Zn(II) complex, steady-state and time-resolved fluorescence studies were performed in ethanol solution and in the solid state. In solution, intramolecular pi-stacking interaction between phenanthroline and anthracene in the ground state and exciplex emission in the excited state were observed. From the temperature dependence of the photostationary ratio (I(Exc)/I(M)), the activation energy for the exciplex formation (E(a)) and the binding energy of the exciplex (-DeltaH) were determined. The crystal structure of the [ZnLBr](ClO(4)).H(2)O compound was resolved, showing that in the solid state both intra- and inter-molecular pi-stacking interactions are present. Such interactions were also evidenced by UV-vis absorption and emission spectra in the solid state. The absorption spectrum of a thin film of the solid complex is red-shifted compared with the solution spectra, whereas its emission spectrum reveals the unique featureless exciplex band, blue shifted compared with the solution. In conjunction with X-ray data the solid-state data was interpreted as being due to a new exciplex where no pi-stacking (full overlap of the pi-electron cloud of the two chromophores - anthracene and phenanthroline) is observed. L is a fluorescent chemosensor able to signal Zn(II) in presence of Cd(II) and Hg(II), since the last two metal ions do not give rise either to the formation of pi-stacking complexes or to exciplex emission in solution. PMID

  5. Zn(II)-coordination modulated ligand photophysical processes – the development of fluorescent indicators for imaging biological Zn(II) ions

    PubMed Central

    Yuan, Zhao; Simmons, J. Tyler; Sreenath, Kesavapillai

    2014-01-01

    Molecular photophysics and metal coordination chemistry are the two fundamental pillars that support the development of fluorescent cation indicators. In this article, we describe how Zn(II)-coordination alters various ligand-centered photophysical processes that are pertinent to developing Zn(II) indicators. The main aim is to show how small organic Zn(II) indicators work under the constraints of specific requirements, including Zn(II) detection range, photophysical requirements such as excitation energy and emission color, temporal and spatial resolutions in a heterogeneous intracellular environment, and fluorescence response selectivity between similar cations such as Zn(II) and Cd(II). In the last section, the biological questions that fluorescent Zn(II) indicators help to answer are described, which have been motivating and challenging this field of research. PMID:25071933

  6. A "turn-on" fluorescent chemosensor for the detection of Zn(II) in aqueous solution at neutral pH and its application in live cells imaging.

    PubMed

    Li, Yuanyuan; Li, Kai; He, Juan

    2016-06-01

    Pyridoxal-2-hydrazinopyridine Schiff-base (1) was prepared by one-step synthesis from commercially available materials of pyridoxal and 2-hydrazinopyridine. Compound 1 could be applied as a fluorescent chemosensor for Zn(II) in aqueous solution at pH 7.4. After binding to Zn(II) with a molar ratio of 1:1, 1 exhibited 17-fold fluorescence enhancement. In addition, 1 showed a good selectivity toward Zn(II) over other metal ions, which can especially distinguish Zn(II) from Cd(II). A linear range of 5.0-20.0μmolL(-1) and a detection limit of 70nmolL(-1) were also achieved. Moreover, the utility of chemosensor 1 in bioimaging of Zn(II) in living cells was demonstrated, which suggested that 1 could be used as a Zn(II)-selective chemosensor in biological samples. PMID:27130131

  7. High-sensitivity determination of Zn(II) and Cu(II) in vitro by fluorescence polarization

    NASA Astrophysics Data System (ADS)

    Thompson, Richard B.; Maliwal, Badri P.; Feliccia, Vincent; Fierke, Carol A.

    1998-04-01

    Recent work has suggested that free Cu(II) may play a role in syndromes such as Crohn's and Wilson's diseases, as well as being a pollutant toxic at low levels to shellfish and sheep. Similarly, Zn(II) has been implicated in some neural damage in the brain resulting from epilepsy and ischemia. Several high sensitivity methods exist for determining these ions in solution, including GFAAS, ICP-MS, ICP-ES, and electrochemical techniques. However, these techniques are generally slow and costly, require pretreatment of the sample, require complex instruments and skilled personnel, and are incapable of imaging at the cellular and subcellular level. To address these shortcomings we developed fluorescence polarization (anisotropy) biosensing methods for these ions which are very sensitivity, highly selective, require simple instrumentation and little pretreatment, and are inexpensive. Thus free Cu(II) or Zn(II) can be determined at picomolar levels by changes in fluorescence polarization, lifetime, or wavelength ratio using these methods; these techniques may be adapted to microscopy.

  8. N,N-Diethylamine appended binuclear Zn(ii) complexes: highly selective and sensitive fluorescent chemosensors for picric acid.

    PubMed

    Kumar, Amit; Kumar, Ashish; Pandey, Daya Shankar

    2016-05-28

    Novel binuclear Zn(ii) complexes (1-2) derived from bis-chelating salen type ligands (H2L(1) and H2L(2)) possessing N,N-diethylamine moieties on the periphery of the molecules have been synthesized and thoroughly characterized by satisfactory elemental analyses and spectral (FT-IR, (1)H, (13)C NMR, UV-vis, fluorescence and ESI-MS) studies. The structures of H2L(1) and 1 have been authenticated by single crystal X-ray diffraction analyses. Complexes 1 and 2 strongly fluoresce and act as highly selective and sensitive chemosensors for picric acid in different organic as well as aqueous media. Both 1 and 2 showed strong potential to detect traces of PA in vapour/solid phase through contact mode analysis. Spectral and theoretical (DFT) studies suggested that the observed fluorescence quenching may be associated with ground state (GS) charge transfer as well as electrostatic interactions between 1/2 and PA. The fluorescence lifetime for the representative complex 1 displayed a double exponential curve and unaltered lifetime (τav, 0.63 nm) in the absence and presence of PA and strongly suggested that quenching follows a static mechanism. Further, DFT calculations on 1 and 2 strongly supported the static mechanism through GS charge transfer between complexes and PA. In addition, (1)H NMR spectral studies on 1-2 in the presence of PA firmly advocated strong hydrogen bonding and π-π stacking between the phenolic rings of 1-2 and the aromatic ring of PA. These complexes are capable of detecting PA either individually or in a competitive environment of other nitro- explosives. Florescence spectral studies on the model complex M lacking N,N-diethylamine groups revealed moderate selectivity and sensitivity towards PA and supported the key role of N,N-diethylamine moieties in the selectivity and sensitivity of complexes. PMID:27114325

  9. Dicynamide bridged two new zig-zag 1-D Zn(II) coordination polymers of pyrimidine derived Schiff base ligands: Synthesis, crystal structures and fluorescence studies

    NASA Astrophysics Data System (ADS)

    Konar, Saugata

    2015-07-01

    Two new zigzag 1-D polymeric Zn(II) coordination polymers {[Zn(L1)(μ1,5-dca)](H2O)}n (1), {[Zn(L2)(μ1,5-dca)](ClO4)}n (2) of two potentially tridentate NNO-, NNN-, donor Schiff base ligands [2-(2-(4,6-dimethylpyrimidin-2-yl)hydrazono)methyl)phenol] (L1), [1-(4,6-dimethylpyrimidin-2-yl)-2-(dipyridin-2ylmethylene)hydrazine] (L2) have been synthesized and characterized by elemental analyses, IR and 1H NMR, fluorescence spectroscopy and single crystal X-ray crystallography. The dicyanamide ions act as linkers (μ1,5 mode) in the formation of these coordination polymers. Both the complexes 1 and 2 have same distorted square pyramidal geometry around the Zn(II) centres. The weak forces like π⋯π, Csbnd H⋯π, anion⋯π interactions lead to various supramolecular architectures. Complex 1 shows high chelation enhanced fluorescence compared to that of 2. The fluorescence spectral changes observed high selectivity towards Zn(II) over other metal ions such as Mn(II), Co(II), Ni(II), Cu(II).

  10. Synthesis, Characterization, and Fluorescence Properties of Mixed Molecular Multilayer Films of BODIPY and Zn(II) Tetraphenylporphyrins.

    PubMed

    Topka, Michael R; Dinolfo, Peter H

    2015-04-22

    A new azido functionalized 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) has been synthesized to achieve spectral complementarity to a Zn(II) tetraphenylethynyl porphyrin (ZnTPEP). Mixed multilayer films were assembled on glass and quartz up to 10 bilayers thick in a layer-by-layer (LbL) fabrication process using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to couple these two dyes together with a tris-azido linker. By varying the amount of BODIPY in the CuAAC reaction solutions for the azido linker layers, we achieve tunable doping of BODIPY within the porphyrin films. We are able to demonstrate linear film growth and determine thickness by X-ray reflectivity (XRR). XRR data indicated that lower BODIPY loading leads to higher porphyrin content and slightly thicker films. Fluorescence emission and excitation spectra of the mixed multilayer films show efficient quenching of the BODIPY singlet and enhanced ZnTPEP emission, suggesting efficient energy transfer (EnT). The ease of fabrication and tunability of these films may serve as potential light harvesting arrays for molecular-based solar cells. PMID:25811793

  11. Fluorescent measurement of affinity binding between thrombin and its aptamers using on-chip affinity monoliths.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Woolley, Adam T

    2013-05-24

    A microfluidic chip with integrated 2mm long monoliths incorporated with poly(ethylene glycol) (PEG) groups was developed for thrombin-aptamer interaction study. The non-G quartet forming oligonucleotide coated monoliths was compared to a 15 mer thrombin-binding aptamer, in which affinity binding and elution processes were real-time monitored fluorescently. The results showed that the fluorescence intensity of aptamer stationary phase is approximately 10 times higher than that of the control column, which is probably due to the successful suppression of nonspecific adsorption between thrombin and aptamers/monoliths by using PEG-monolith. The experiment was repeated using human serum albumin (HSA) and green fluorescence protein (GFP) as interferences, it was double confirmed that thrombin was selectively retained by PEG-monolith. An elution efficiency of 75% was achieved with an elute of 200mM acetic acid and 2M NaCI, and the eluted thrombin was successfully separated in an ionic buffer system of 20mM NaHCO3 (pH 9.5) with 3% PEG. The hydrophilic and antifouling properties of PEG-monolith greatly decrease nonspecific adsorption and enhance detection sensitivity, which provided an alternative method to perform on-chip fluorescent measurement of bioaffinity binding. PMID:23587316

  12. High kinetic stability of Zn(II) coordinated by the tris(histidine) unit of carbonic anhydrase towards solvolytic dissociation studied by affinity capillary electrophoresis.

    PubMed

    Sato, Yosuke; Hoshino, Hitoshi; Iki, Nobuhiko

    2016-08-01

    Solvolytic dissociation rate constants (kd) of bovine carbonic anhydrase II (CA) and its metallovariants (M-CAs, M=Co(II), Ni(II), Cu(II), Zn(II), and Cd(II)) were estimated by a ligand substitution reaction, which was monitored by affinity capillary electrophoresis to selectively detect the undissociated CAs in the reaction mixture. Using EDTA as the competing ligand for Zn-CA, the dissociation followed the unimolecular nucleophilic substitution (SN1) mechanism with kd=1.0×10(-7)s(-1) (pH7.4, 25°C). The corresponding solvolysis half-life (t1/2) was 80days, showing the exceptionally high kinetic stability of t Zn-CA, in contrast to the highly labile [Zn(II)(H2O)6](2+), where the water exchange rate (kex) is high. This behavior is attributed to the tetrahedral coordination geometry supported by the tris(histidine) unit (His3) of CA. In the case of Co-CA, it showed a somewhat larger kd value (5.7×10(-7)s(-1), pH7.4, 25°C) even though it shares the same tetrahedral coordination environment with Zn-CA, suggesting that the d(7) electronic configuration of Co(II) in the transition state of the dissociation is stabilized by the ligand field. Among M-CAs, only Ni-CA showed a bimolecular nucleophilic substitution (SN2) reaction path in its reaction with EDTA, implying that the large coordination number (6) of Ni(II) in Ni-CA allows EDTA to form an EDTA-Ni-CA intermediate. Overall, kd values roughly correlated with kex values among M-CAs, with the kd value of Zn-CA deviating strongly from the trend and highlighting the exceptionally high kinetic stabilization of Zn-CA by the His3 unit. PMID:27235274

  13. Nanoscale {LnIII(24)ZnII(6)} Triangular Metalloring with Magnetic Refrigerant, Slow Magnetic Relaxation, and Fluorescent Properties.

    PubMed

    Zhang, Li; Zhao, Lang; Zhang, Peng; Wang, Chao; Yuan, Sen-Wen; Tang, Jinkui

    2015-12-01

    The self-assembly of Ln(ClO4)3 · 6H2O and Zn(OAc)2 · 2H2O with pyrazine-2-carboxylic acid (HL) results in the formation of three novel nanosized {LnIII(24)ZnII(6)} triangular metallorings, [Gd24Zn6L24(OAc)22(μ3-OH)30(H2O)14](ClO4)7(OAc) · 2CH3OH · 26H2O (1), [Tb24Zn6L24(OAc)22(μ3-OH)30(CH3O)2(CH3OH)2(H2O)10](ClO4)5(OH) · 6CH3OH · 12H2O (2), and (H3O)[Dy24Zn6L24(OAc)22(μ3-OH)30(H2O)14](ClO4)7(OAc)2 · 4CH3OH · 22H2O (3), having the largest nuclearity among any known Ln/Zn clusters. Magnetic and luminescent studies reveal the special prowess for each lanthanide complex. Magnetic studies reveal that 1 exhibits a significant cryogenic magnetocaloric effect with a maximum -ΔSm (isothermal magnetic entropy change) value of 30.0 J kg(-1) K(-1) at 2.5 K and 7 T and that a slow magnetization relaxation is observed for the dysprosium analogue. In addition, the solid-state photophysical properties of 2 display strong characteristic Tb(III) photoluminescent emission in the visible region, suggesting that Tb(III)-based luminescence is sensitized by the effective energy transfer from the ligand HL to the metal centers. PMID:26600284

  14. Bodilisant—A Novel Fluorescent, Highly Affine Histamine H3 Receptor Ligand

    PubMed Central

    2012-01-01

    A piperidine-based lead structure for the human histamine H3 receptor (hH3R) was coupled with the BODIPY fluorophore and resulted in a strong green fluorescent (quantum yield, 0.92) hH3R ligand with affinity in the nanomolar concentration range (Ki hH3R = 6.51 ± 3.31 nM), named Bodilisant. Screening for affinities at histamine and dopamine receptor subtypes showed high hH3R preference. Bodilisant was used for visualization of hH3R in hH3R overexpressing HEK-293 cells with fluorescence confocal laser scanning microscopy. In addition, in native human brain tissues, Bodilisant showed clear and displaceable images of labeled hH3R. PMID:24900647

  15. Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

    PubMed Central

    Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

    2016-01-01

    The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096

  16. Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions.

    PubMed

    Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

    2016-01-01

    The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. PMID:27436096

  17. Fluorescent Boronic Acid Polymer Grafted on Silica Particles for Affinity Separation of Saccharides

    PubMed Central

    2014-01-01

    Boronic acid affinity gels are important for effective separation of biological active cis-diols, and are finding applications both in biotech industry and in biomedical research areas. To increase the efficacy of boronate affinity separation, it is interesting to introduce repeating boronic acid units in flexible polymer chains attached on solid materials. In this work, we synthesize polymer brushes containing boronic acid repeating units on silica gels using surface-initiated atom transfer radical polymerization (ATRP). A fluorescent boronic acid monomer is first prepared from an azide-tagged fluorogenic boronic acid and an alkyne-containing acrylate by Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction (the CuAAC click chemistry). The boronic acid monomer is then grafted to the surface of silica gel modified with an ATRP initiator. The obtained composite material contains boronic acid polymer brushes on surface and shows favorable saccharide binding capability under physiological pH conditions, and displays interesting fluorescence intensity change upon binding fructose and glucose. In addition to saccharide binding, the flexible polymer brushes on silica also enable fast separation of a model glycoprotein based on selective boronate affinity interaction. The synthetic approach and the composite functional material developed in this work should open new opportunities for high efficiency detection, separation, and analysis of not only simple saccharides, but also glycopeptides and large glycoproteins. PMID:24444898

  18. Fluorescent boronic acid polymer grafted on silica particles for affinity separation of saccharides.

    PubMed

    Xu, Zhifeng; Uddin, Khan Mohammad Ahsan; Kamra, Tripta; Schnadt, Joachim; Ye, Lei

    2014-02-12

    Boronic acid affinity gels are important for effective separation of biological active cis-diols, and are finding applications both in biotech industry and in biomedical research areas. To increase the efficacy of boronate affinity separation, it is interesting to introduce repeating boronic acid units in flexible polymer chains attached on solid materials. In this work, we synthesize polymer brushes containing boronic acid repeating units on silica gels using surface-initiated atom transfer radical polymerization (ATRP). A fluorescent boronic acid monomer is first prepared from an azide-tagged fluorogenic boronic acid and an alkyne-containing acrylate by Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction (the CuAAC click chemistry). The boronic acid monomer is then grafted to the surface of silica gel modified with an ATRP initiator. The obtained composite material contains boronic acid polymer brushes on surface and shows favorable saccharide binding capability under physiological pH conditions, and displays interesting fluorescence intensity change upon binding fructose and glucose. In addition to saccharide binding, the flexible polymer brushes on silica also enable fast separation of a model glycoprotein based on selective boronate affinity interaction. The synthetic approach and the composite functional material developed in this work should open new opportunities for high efficiency detection, separation, and analysis of not only simple saccharides, but also glycopeptides and large glycoproteins. PMID:24444898

  19. Sorption of Zn(II) in aqueous solutions by scoria.

    PubMed

    Kwon, Jang-Soon; Yun, Seong-Taek; Kim, Soon-Oh; Mayer, Bernhard; Hutcheon, Ian

    2005-09-01

    We conducted kinetic and equilibrium sorption experiments on removal of Zn(II) from aqueous solutions by scoria (a vesicular pyroclastic rock with basaltic composition) from Jeju Island, Korea, in order to examine its potential use as an efficient sorbent. The batch-type kinetic sorption tests under variable conditions indicated that the percentage of Zn(II) removal by scoria increases with decreasing initial Zn(II) concentration, particle size, and sorbate/sorbent ratio. However, the sorption capacity decreases with the decrease of the initial Zn(II) concentration and sorbate/sorbent ratio. Equilibrium sorption tests show that Jeju scoria has a larger capacity and affinity for Zn(II) sorption than commercial powdered activated carbon (PAC); at initial Zn(II) concentrations of more than 10mM, the sorption capacity of Jeju scoria is about 1.5 times higher than that of PAC. The acquired sorption data are better fitted to the Langmuir isotherm than the Freundlich isotherm. Careful examination of ionic concentrations in sorption batches suggests that the sorption behavior is mainly controlled by cation exchange and typically displays characteristics of 'cation sorption'. The Zn(II) removal capacity decreases when solution pH decreases because of the competition with hydrogen ions for sorption sites, while the Zn(II) removal capacity increases under higher pH conditions, likely due to hydroxide precipitation. At an initial Zn(II) concentration of 5.0mM, the removal increases from 70% to 96% with the increase of initial pH from 3.0 to 7.0. We recommend Jeju scoria as an economic and efficient sorbent for Zn(II) in contaminated water. PMID:16054911

  20. Fluorescence Characterization of Dissolved Organic Matter Isolates from Sediments and the Association with Phenanthrene Binding Affinity

    NASA Astrophysics Data System (ADS)

    Hur, Jin; Lee, Bo-Mi; Shin, Kyung-Hoon

    2014-05-01

    In this study, selected spectroscopic characteristics of sediment organic matter (SOM) were compared and discussed with respect to their different isolation methods, the source discrimination capabilities, and the association with the extent of phenanthrene binding. A total of 16 sediments were collected from three categorized locations including a costal lake, industrial areas, and the upper streams, each of which is likely influenced by the organic sources of algal production, industrial effluent, and terrestrial input, respectively. The spectroscopic properties related to aromatic structures and terrestrial humic acids were more pronounced for alkaline extractable organic matter (AEOM) isolates than for the SOM isolates based on water soluble extracts and porewater. The three categorized sampling locations were the most differentiated in the AEOM isolates, suggesting AEOM may be the most representative SOM isolates in describing the chemical properties and the organic sources of SOM. Parallel factor analysis (PARAFAC) based on fluorescence excitation-emission matrix (EEM) showed that a combination of four fluorescent groups could represent all the fluorescence features of SOM. The three categorized sampling locations were well discriminated by the percent distributions of terrestrial and microbial humic-like fluorescent groups of the AEOM isolates. The relative distribution of terrestrial humic-like fluorophores was highly correlated with the extent of phenanthrene binding (r=0.676; p<0.01), suggesting that the presence of terrestrial humic acids in SOM may contribute to the enhancement of binding with hydrophobic organic contaminants in sediments. Principal component analysis (PCA) further demonstrated that the extent of SOM's binding affinity might be affected by the degree of biological transformation in SOM as well as the abundance of aromatic carbon structures.

  1. A High Affinity Red Fluorescence and Colorimetric Probe for Amyloid β Aggregates

    NASA Astrophysics Data System (ADS)

    Rajasekhar, K.; Narayanaswamy, Nagarjun; Murugan, N. Arul; Kuang, Guanglin; Ågren, Hans; Govindaraju, T.

    2016-04-01

    A major challenge in the Alzheimer’s disease (AD) is its timely diagnosis. Amyloid β (Aβ) aggregates have been proposed as the most viable biomarker for the diagnosis of AD. Here, we demonstrate hemicyanine-based benzothiazole-coumarin (TC) as a potential probe for the detection of highly toxic Aβ42 aggregates through switch-on, enhanced (~30 fold) red fluorescence (Emax = 654 nm) and characteristic colorimetric (light red to purple) optical outputs. Interestingly, TC exhibits selectivity towards Aβ42 fibrils compared to other abnormal protein aggregates. TC probe show nanomolar binding affinity (Ka = 1.72 × 107 M‑1) towards Aβ42 aggregates and also displace ThT bound to Aβ42 fibrils due to its high binding affinity. The Aβ42 fibril-specific red-shift in the absorption spectra of TC responsible for the observed colorimetric optical output has been attributed to micro-environment change around the probe from hydrophilic-like to hydrophobic-like nature. The binding site, binding energy and changes in optical properties observed for TC upon interaction with Aβ42 fibrils have been further validated by molecular docking and time dependent density functional theory studies.

  2. A High Affinity Red Fluorescence and Colorimetric Probe for Amyloid β Aggregates.

    PubMed

    Rajasekhar, K; Narayanaswamy, Nagarjun; Murugan, N Arul; Kuang, Guanglin; Ågren, Hans; Govindaraju, T

    2016-01-01

    A major challenge in the Alzheimer's disease (AD) is its timely diagnosis. Amyloid β (Aβ) aggregates have been proposed as the most viable biomarker for the diagnosis of AD. Here, we demonstrate hemicyanine-based benzothiazole-coumarin (TC) as a potential probe for the detection of highly toxic Aβ42 aggregates through switch-on, enhanced (~30 fold) red fluorescence (Emax = 654 nm) and characteristic colorimetric (light red to purple) optical outputs. Interestingly, TC exhibits selectivity towards Aβ42 fibrils compared to other abnormal protein aggregates. TC probe show nanomolar binding affinity (Ka = 1.72 × 10(7) M(-1)) towards Aβ42 aggregates and also displace ThT bound to Aβ42 fibrils due to its high binding affinity. The Aβ42 fibril-specific red-shift in the absorption spectra of TC responsible for the observed colorimetric optical output has been attributed to micro-environment change around the probe from hydrophilic-like to hydrophobic-like nature. The binding site, binding energy and changes in optical properties observed for TC upon interaction with Aβ42 fibrils have been further validated by molecular docking and time dependent density functional theory studies. PMID:27032526

  3. A High Affinity Red Fluorescence and Colorimetric Probe for Amyloid β Aggregates

    PubMed Central

    Rajasekhar, K.; Narayanaswamy, Nagarjun; Murugan, N. Arul; Kuang, Guanglin; Ågren, Hans; Govindaraju, T.

    2016-01-01

    A major challenge in the Alzheimer’s disease (AD) is its timely diagnosis. Amyloid β (Aβ) aggregates have been proposed as the most viable biomarker for the diagnosis of AD. Here, we demonstrate hemicyanine-based benzothiazole-coumarin (TC) as a potential probe for the detection of highly toxic Aβ42 aggregates through switch-on, enhanced (~30 fold) red fluorescence (Emax = 654 nm) and characteristic colorimetric (light red to purple) optical outputs. Interestingly, TC exhibits selectivity towards Aβ42 fibrils compared to other abnormal protein aggregates. TC probe show nanomolar binding affinity (Ka = 1.72 × 107 M−1) towards Aβ42 aggregates and also displace ThT bound to Aβ42 fibrils due to its high binding affinity. The Aβ42 fibril-specific red-shift in the absorption spectra of TC responsible for the observed colorimetric optical output has been attributed to micro-environment change around the probe from hydrophilic-like to hydrophobic-like nature. The binding site, binding energy and changes in optical properties observed for TC upon interaction with Aβ42 fibrils have been further validated by molecular docking and time dependent density functional theory studies. PMID:27032526

  4. Affinity fluorescence-labeled peptides for the early detection of cancer in Barrett's esophagus

    NASA Astrophysics Data System (ADS)

    Li, Meng; Lu, Shaoying; Piraka, Cyrus; Appelman, Henry; Kwon, Rich; Soetikno, Roy; Kaltenbach, Tonya; Wang, Thomas D.

    2009-02-01

    Fluorescence-labeled peptides that affinity bind to neoplastic mucsosa are promising for use as a specific contrast agent in the detection of pre-malignant tissue in the esophagus. This method is can be used to identify expression of biological markers associated with dysplasia on endoscopic imaging as a guide for biopsy and represents a novel method for the early detection and prevention of cancer. We demonstrate the use of phage display to select affinity peptides and identify the sequence "ASYNYDA" that binds with high target-to-background ratio to dysplastic esophageal mucosa compared to that of intestinal metaplasia. Validation of preferential binding is demonstrated for neoplasia in the setting of Barrett's esophagus. An optimal tradeoff between sensitivity and specificity of 82% and 85% was found at the relative threshold of 0.60 with a target-to-background ratio of 1.81 and an area under the ROC curve of 0.87. Peptides are a novel class of ligand for targeted detection of pre-malignant mucosa for purposes of screening and surveillance.

  5. An Affinity-Based Fluorescence Polarization Assay for Protein Tyrosine Phosphatases

    PubMed Central

    Zhang, Sheng; Chen, Lan; Kumar, Sanjai; Wu, Li; Lawrence, David S.; Zhang, Zhong-Yin

    2007-01-01

    Protein tyrosine phosphatases (PTPs) are important signaling enzymes that control such fundamental processes as proliferation, differentiation, survival/apoptosis, as well as adhesion and motility. Potent and selective PTP inhibitors serve not only as powerful research tools, but also as potential therapeutics against a variety illness including cancer and diabetes. PTP activity-based assays are widely used in high throughput screening (HTS) campaigns for PTP inhibitor discovery. These assays suffer from a major weakness, in that the reactivity of the active site Cys can cause serious problems as highly reactive oxidizing and alkylating agents may surface as hits. We describe the development of a fluorescence polarization (FP)-based displacement assay that makes the use of an active site Cys to Ser mutant PTP (e.g., PTP1B/C215S) that retains the wild type binding affinity. The potency of library compounds is assessed by their ability to compete with the fluorescently labeled active site ligand for binding to the Cys to Ser PTP mutant. Finally, the substitution of the active site Cys by a Ser renders the mutant PTP insensitive to oxidation and alkylation and thus will likely eliminate “false” positives due to modification of the active site Cys that destroy the phosphatase activity. PMID:17532513

  6. Monitoring water supplies for weaponized bacteria and bacterial toxins using rapid fluorescence-based viability and affinity assays

    NASA Astrophysics Data System (ADS)

    Van Tassell, Roger L.; Evans, Mishell

    2004-03-01

    The rapid detection of weaponized bacteria and toxins is a major problem during a biological attack. Although sensitive detection formats exist for many biowarfare agents, they often require advanced training and complex procedures. Luna has developed simple, rapid means for determining the presence of pathogens and bacterial toxins in water supplies using fluorescence-based assays that can be adapted for field use. The batteries of rapid assays are designed for i) determining cell viability and bacterial loads by exploiting metabolic markers (e.g., acid-production, redox potentials, etc) and ii) detecting bacterial toxins using fluorescent, polymerized affinity liposomes (fluorosomes). The viability assays were characterized using E. coli, S. aureus and the anthrax simulant, B. globigii. The viability assays detected bacterial loads of ~ 104 CFU/ml and with simple filtration ~ 100CFU/ml could be detected. The affinity fluorosomes were characterized using cholera toxin (CT). Affinity liposomes displaying GM1 and anti-CT antibodies could detect CT at <μg/ml levels. Stability studies showed that affinity vesicles could be stored for weeks at 4°C or freeze-dried with no significant loss of binding capacity. Using an in-house fiber optic fluorescence system, Luna characterized the binding of affinity fluorosomes to respective targets and determined the responses of bacterial loads in the fluorescent viability assays. Using this two-tiered approach, Luna demonstrated that water susceptible to sabotage could be easily monitored and confirmed for specific agents using simple, general and specific fluorescence-based detection schemes based on metabolism and ligand-target interactions.

  7. Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current

    NASA Astrophysics Data System (ADS)

    Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro

    2011-10-01

    Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

  8. Development of BODIPY FL Vindoline as a Novel and High-Affinity Pregnane X Receptor Fluorescent Probe

    PubMed Central

    2015-01-01

    The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows it to bind many drugs and drug leads, possibly causing undesired drug–drug interactions. Therefore, it is crucial to evaluate whether chemicals or drugs bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and nonradioactive nature. On the basis of our previously characterized 4 (BODIPY FL vinblastine), a high-affinity PXR probe, we developed 20 (BODIPY FL vindoline) and showed that it is a novel and potent PXR fluorescent probe with Kd of 256 nM in a time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay with PXR. By using 20 (BODIPY FL vindoline) in the PXR TR-FRET assay, we obtained a more than 7-fold signal-to-background ratio and high signal stability (signal was stable for at least 120 min, and Z′-factor > 0.85 from 30 to 240 min). The assay can tolerate DMSO up to 2%. This assay has been used to evaluate a panel of PXR ligands for their PXR-binding affinities. The performance of 20 (BODIPY FL vindoline) in the PXR TR-FRET assay makes it an ideal PXR fluorescent probe, and the newly developed PXR TR-FRET assay with 20 (BODIPY FL vindoline) as a fluorescent probe is suitable for high-throughput screening to identify PXR-binding ligands. PMID:25133934

  9. Synthesis of a Targeted Biarsenical Cy3-Cy5 Affinity Probe for Superresolution Fluorescence Imaging

    SciTech Connect

    Fu, Na; Xiong, Yijia; Squier, Thomas C.

    2012-11-01

    Photoswitchable fluorescent probes capable of the targeted labeling of tagged proteins are of significant interest due to their ability to enable in situ imaging of protein complexes within native biomolecular assemblies. Here we describe the synthesis of a fluorescent probe (AsCy3Cy5), and demonstrate the targeted labeling and super-resolution imaging of a tagged protein within a supramolecular protein complex.

  10. ICPBCZin: a red emitting ratiometric fluorescent indicator with nanomolar affinity for Zn2+ ions.

    PubMed

    Roussakis, Emmanuel; Voutsadaki, Styliani; Pinakoulaki, Eftychia; Sideris, Dionisia P; Tokatlidis, Kostas; Katerinopoulos, Haralambos E

    2008-09-01

    A new fluorescent Zn2+ indicator, namely, ICPBCZin was synthesized and the spectral profile of its free and Zn2+ bound forms was studied. The newly synthesized zinc indicator incorporates as chromophore the chromeno [3',2':3,4]pyrido[1,2a] [1,3]benzimidazole moiety and belongs to the dicarboxylate-type of zinc probes. The compound is excited with visible light, exhibits high selectivity for zinc in the presence of calcium and other common biological ions, and its Zn2+ dissociation constant is 4.0 nM. Fluorescence spectra studies of ICPBCZin indicated a clear shift in its emission wavelength maxima upon Zn2+ binding, as it belongs to the class of Photoinduced Charge Transfer (PCT) indicators, along with changes in fluorescence intensity that enable the compound to be used as a ratiometric, visible-excitable Zn2+ probe. PMID:18243303

  11. Microtubule Affinity Regulating Kinase Activity in Living Neurons Was Examined by a Genetically Encoded Fluorescence Resonance Energy Transfer/Fluorescence Lifetime Imaging-based Biosensor

    PubMed Central

    Timm, Thomas; von Kries, Jens Peter; Li, Xiaoyu; Zempel, Hans; Mandelkow, Eckhard; Mandelkow, Eva-Maria

    2011-01-01

    Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ∼18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons. PMID:21984823

  12. Boronate Affinity Fluorescent Nanoparticles for Förster Resonance Energy Transfer Inhibition Assay of cis-Diol Biomolecules.

    PubMed

    Wang, Shuangshou; Ye, Jin; Li, Xinglin; Liu, Zhen

    2016-05-17

    Förster resonance energy transfer (FRET) has been essential for many applications, in which an appropriate donor-acceptor pair is the key. Traditional dye-to-dye combinations remain the working horses but are rather nonspecifically susceptive to environmental factors (such as ionic strength, pH, oxygen, etc.). Besides, to obtain desired selectivity, functionalization of the donor or acceptor is essential but usually tedious. Herein, we present fluorescent poly(m-aminophenylboronic acid) nanoparticles (poly(mAPBA) NPs) synthesized via a simple procedure and demonstrate a FRET scheme with suppressed environmental effects for the selective sensing of cis-diol biomolecules. The NPs exhibited stable fluorescence properties, resistance to environmental factors, and a Förster distance comparable size, making them ideal donor for FRET applications. By using poly(mAPBA) NPs and adenosine 5'-monophosphate modified graphene oxide (AMP-GO) as a donor and an acceptor, respectively, an environmental effects-suppressed boronate affinity-mediated FRET system was established. The fluorescence of poly(mAPBA) NPs was quenched by AMP-GO while it was restored when a competing cis-diol compounds was present. The FRET system exhibited excellent selectivity and improved sensitivity toward cis-diol compounds. Quantitative inhibition assay of glucose in human serum was demonstrated. As many cis-diol compounds such as sugars and glycoproteins are biologically and clinically significant, the FRET scheme presented herein could find more promising applications. PMID:27089186

  13. Rapid Macrocycle Threading by a Fluorescent Dye-Polymer Conjugate in Water with Nanomolar Affinity

    PubMed Central

    Peck, Evan M.; Liu, Wenqi; Spence, Graeme T.; Shaw, Scott K.; Davis, Anthony P.; Destecroix, Harry; Smith, Bradley D.

    2015-01-01

    A macrocyclic tetralactam host is threaded by a highly fluorescent squaraine dye that is flanked by two polyethyleneglycol (PEG) chains with nanomolar dissociation constants in water. Furthermore, the rates of bimolecular association are very fast with kon ~106–107 M−1s−1. The association is effective under cell culture conditions and produces large changes in dye optical properties including turn-on near-infrared fluorescence that can be imaged using cell microscopy. Association constants in water are ~1000 times higher than in organic solvents and strongly enthalpically favored at 27 °C. The threading rate is hardly affected by the length of the PEG chains that flank the squaraine dye. For example, macrocyle threading by a dye conjugate with two appended PEG2000 chains is only three times slower than threading by a conjugate with triethyleneglycol chains that are twenty times shorter. The results are a promising advance towards synthetic mimics of streptavidin/biotin. PMID:26106948

  14. Fluorescence microscopy studies with a fluorescent glibenclamide derivative, a high-affinity blocker of pancreatic beta-cell ATP-sensitive K+ currents.

    PubMed

    Zünkler, Bernd J; Wos-Maganga, Maria; Panten, Uwe

    2004-04-15

    Hypoglycemic sulfonylureas (e.g. tolbutamide, glibenclamide) exert their stimulatory effects on pancreatic beta-cells by closure of ATP-sensitive K(+) (K(ATP)) channels. Pancreatic K(ATP) channels are composed of two subunits, a pore-forming inwardly rectifying K(+) channel (Kir6.2) subunit and a regulatory subunit (the sulfonylurea receptor of subtype 1 (SUR1)) in a (SUR1/Kir6.2)(4) stoichiometry. The aim of the present study was to characterize the interaction of green-fluorescent 3-[3-(4,4 difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacen-3-yl)propanamido] glibenclamide (Bodipy-glibenclamide) with pancreatic beta-cell K(ATP) channels using patch-clamp and fluorescence microscopy techniques. Bodipy-glibenclamide inhibited K(ATP) currents from the clonal insulinoma cell line RINm5F half-maximally at a concentration of 0.6nM. Using laser-scanning confocal microscopy Bodipy-glibenclamide was shown to induce a diffuse fluorescence across the RINm5F cell, but only about 17% of total Bodipy-glibenclamide-induced fluorescence intensity in RINm5F cells was due to specific binding to SUR1. Using fluorescence correlation spectroscopy, it could be demonstrated that the fluorescence label contributes to the protein binding and, therefore, possibly also to the non-specific binding of Bodipy-glibenclamide observed in RINm5F cells. Specific binding of Bodipy-glibenclamide to SUR1 in RINm5F cells might be localized to different intracellular structures (nuclear envelope, endoplasmic reticulum, Golgi compartment, insulin secretory granules) as well as to the plasma membrane. In conclusion, Bodipy-glibenclamide is a high-affinity blocker of pancreatic beta-cell K(ATP) currents and can be used for visualizing SUR1 in intact pancreatic beta-cells, although non-specific binding must be taken into account in confocal microscopy experiments on intact beta-cells. PMID:15041461

  15. Immobilized metal ion affinity-based fluorescence polarization (IMAP): advances in kinase screening.

    PubMed

    Sportsman, J Richard; Gaudet, Elizabeth A; Boge, Annegret

    2004-04-01

    The IMAP Fluorescence Polarization technology is a homogeneous antibody-free method for analysis of kinases, phosphatases, and phosphodiesterases. Recent developments to the technology include an enhancement of the reagent system (the Progressive Binding System) that significantly expands the range of useable concentrations of ATP, choices of substrates, and assay configurations. With the new Progressive System, we are able to design multiplexed assays that allow the simultaneous determination of multiple kinase activities. In addition, coupled assays are now possible, allowing the assay of kinases through natural or artificial coupling through kinase cascades. PMID:15165516

  16. Rapid Macrocycle Threading by a Fluorescent Dye-Polymer Conjugate in Water with Nanomolar Affinity.

    PubMed

    Peck, Evan M; Liu, Wenqi; Spence, Graeme T; Shaw, Scott K; Davis, Anthony P; Destecroix, Harry; Smith, Bradley D

    2015-07-15

    A macrocyclic tetralactam host is threaded by a highly fluorescent squaraine dye that is flanked by two polyethylene glycol (PEG) chains with nanomolar dissociation constants in water. Furthermore, the rates of bimolecular association are very fast with k(on) ≈ 10(6)-10(7) M(-1) s(-1). The association is effective under cell culture conditions and produces large changes in dye optical properties including turn-on near-infrared fluorescence that can be imaged using cell microscopy. Association constants in water are ∼1000 times higher than those in organic solvents and strongly enthalpically favored at 27 °C. The threading rate is hardly affected by the length of the PEG chains that flank the squaraine dye. For example, macrocycle threading by a dye conjugate with two appended PEG2000 chains is only three times slower than threading by a conjugate with triethylene glycol chains that are 20 times shorter. The results are a promising advance toward synthetic mimics of streptavidin/biotin. PMID:26106948

  17. Visualizing Microtubule-Dependent Vasopressin Type 2 Receptor Trafficking Using a New High-Affinity Fluorescent Vasopressin Ligand

    PubMed Central

    Chen, Sylvia; Webber, Matthew J.; Vilardaga, Jean-Pierre; Khatri, Ashok; Brown, Dennis; Ausiello, Dennis A.; Lin, Herbert Y.

    2011-01-01

    The vasopressin receptor type 2 (V2R) is the major target of vasopressin (VP) in renal epithelial cells. Although it is known that VP induces V2R internalization, accumulation in the perinuclear area, and degradation, the V2R intracellular trafficking pathways remain elusive. We visualized this process by developing a new fluorescent VP analog tagged by tetramethylrhodamine (TMR)-[Lys-(PEG)2-Suc-TMR8]VP or (VPTMR). This ligand is fully functional as revealed by its high binding affinity toward V2R [(Kd) =157 ± 52 nm] and ability to increase intracellular cAMP 32-fold. VPTMR induced V2R internalization in LLC-PK1 cells expressing either a FLAG-tagged receptor (FLAG-V2R) or V2R C-terminally tagged with green fluorescent protein (GFP) (V2R-GFP). After internalization, VPTMR and V2R-GFP colocalized in the perinuclear area, suggesting that the hormone and receptor traffic along the same pathway. VPTMR and V2R colocalized initially with the early endosome markers EEA1 and Rab5, and later with the recycling and late endosome markers Rab11 and Rab25. Epifluorescence microscopy of LLC-PK1 cells expressing GFP-tagged microtubules (MT) showed that VPTMR-containing vesicles travel along the MT network, and even remain attached to MT during the metaphase and anaphase of mitosis. Colchicine, a MT-depolymerizing agent, abolished perinuclear accumulation of VPTMR, and Western blot analysis showed that VP-induced V2R-GFP degradation is markedly retarded, but not abolished, by colchicine (10 μM). We conclude that the new VPTMR ligand is suitable for dissecting V2R and VP internalization and trafficking in cells, and that V2R trafficking and down-regulation is an MT-dependent mechanism. PMID:21828182

  18. A set of robust fluorescent peptide probes for quantification of Cu(ii) binding affinities in the micromolar to femtomolar range.

    PubMed

    Young, Tessa R; Wijekoon, Chathuri J K; Spyrou, Benjamin; Donnelly, Paul S; Wedd, Anthony G; Xiao, Zhiguang

    2015-03-01

    Reliable quantification of copper binding affinities and identification of the binding sites provide a molecular basis for an understanding of the nutritional roles and toxic effects of copper ions. Sets of chromophoric probes are now available that can quantify Cu(i) binding affinities from nanomolar to attomolar concentrations on a unified scale under in vitro conditions. Equivalent probes for Cu(ii) are lacking. This work reports development of a set of four fluorescent dansyl peptide probes (DP1-4) that can quantify Cu(ii) binding affinities from micromolar to femtomolar concentrations, also on a unified scale. The probes were constructed by conjugation of a dansyl group to four short peptides of specific design. Each was characterised by its dissociation constant KD, its pH dependence and the nature of its binding site. One equivalent of Cu(ii) is bound by the individual probes that display different and well-separated affinities at pH 7.4 (log KD = -8.1, -10.1, -12.3 and -14.1, respectively). Intense fluorescence is emitted at λmax ∼ 550 nm upon excitation at ∼330 nm. Binding of Cu(ii) quenches the fluorescence intensity linearly until one equivalent of Cu(ii) is bound. Multiple approaches and multiple affinity standards were employed to ensure reliability. Selected examples of application to well-characterised Cu(ii) binding peptides and proteins are presented. These include Aβ16 peptides, two naturally occurring Cu(ii)-chelating motifs in human serum and cerebrospinal fluid with sequences GHK and DAHK and two copper binding proteins, CopC from Pseudomonas syringae and PcoC from Escherichia coli. Previously reported affinities are reproduced, demonstrating that peptides DP1-4 form a set of robust and reliable probes for Cu(ii) binding to peptides and protein targets. PMID:25715324

  19. Effect of biofilm coatings at metal-oxide/water interfaces I: Pb(II) and Zn(II) partitioning and speciation at Shewanella oneidensis/metal-oxide/water interfaces

    NASA Astrophysics Data System (ADS)

    Wang, Yingge; Gélabert, Alexandre; Michel, F. Marc; Choi, Yongseong; Gescher, Johannes; Ona-Nguema, Georges; Eng, Peter J.; Bargar, John R.; Farges, Francois; Spormann, Alfred M.; Brown, Gordon E.

    2016-09-01

    Microbial biofilms are often present as coatings on metal-oxide surfaces in natural and industrial environments and may induce significant changes in the partitioning behavior and speciation of aqueous metal ions, which in turn can impact their transport and fate. In this study, long-period X-ray standing wave-fluorescence yield (LP-XSW-FY) spectroscopy was used to measure under in situ conditions the partitioning of aqueous Pb(II) and Zn(II) between multilayer Shewanella oneidensis MR-1 biofilms and highly polished, oriented single-crystal surfaces of α-Al2O3 and α-Fe2O3 as a function of metal-ion concentration and time at pH 6.0. We show that after 3-h exposure time, Pb(II) binds preferentially to the α-Al2O3 (1-102) and α-Fe2O3 (0 0 0 1) surfaces at low Pb concentration ([Pb] = 10-7 M) and then increasingly partitions into the biofilm coatings at higher concentrations (10-6 to 10-4 M). In contrast, Zn(II) partitions preferentially into the biofilm coating for both surfaces at all Zn concentrations studied (10-7 to 10-4 M). In comparison, the α-Al2O3 (0 0 0 1) surface has a low affinity for both Pb(II) and Zn(II), and the biofilm coatings are the dominant sink for both ions. These findings suggest that in the presence of S. oneidensis biofilm coatings, α-Al2O3 (0 0 0 1) is the least reactive surface for Pb(II) and Zn(II) compared to α-Al2O3 (1-102) and α-Fe2O3 (0 0 0 1). They also show that Zn(II) has a lower affinity than Pb(II) for reactive sites on α-Al2O3 (1-102) and α-Fe2O3 (0 0 0 1) at [Me(II)] of 10-7 M; at 10-5 M, the bulk of the metal ions partition into the biofilm coatings. At longer exposure times (20-24 h), both Pb(II) and Zn(II) increasingly partition to the metal-oxide surfaces at [Me(II)] = 10-5 M and pH 6.0, indicating possible reaction/diffusion-controlled sorption processes. Pb LIII-edge and Zn K-edge grazing-incidence extended X-ray absorption fine structure (GI-EXAFS) measurements suggest that both Pb(II) and Zn(II) ions may be

  20. Role of Bound Zn(II) in the CadC Cd(II)/Pb(II)/Zn(II)-Responsive Repressor

    SciTech Connect

    Kandegedara, A.; Thiyagarajan, S; Kondapalli, K; Stemmler, T; Rosen, B

    2009-01-01

    The Staphylococcus aureus plasmid pI258 cadCA operon encodes a P-type ATPase, CadA, that confers resistance to Cd(II)/Pb(II)/Zn(II). Expression is regulated by CadC, a homodimeric repressor that dissociates from the cad operator/promoter upon binding of Cd(II), Pb(II), or Zn(II). CadC is a member of the ArsR/SmtB family of metalloregulatory proteins. The crystal structure of CadC shows two types of metal binding sites, termed Site 1 and Site 2, and the homodimer has two of each. Site 1 is the physiological inducer binding site. The two Site 2 metal binding sites are formed at the dimerization interface. Site 2 is not regulatory in CadC but is regulatory in the homologue SmtB. Here the role of each site was investigated by mutagenesis. Both sites bind either Cd(II) or Zn(II). However, Site 1 has higher affinity for Cd(II) over Zn(II), and Site 2 prefers Zn(II) over Cd(II). Site 2 is not required for either derepression or dimerization. The crystal structure of the wild type with bound Zn(II) and of a mutant lacking Site 2 was compared with the SmtB structure with and without bound Zn(II). We propose that an arginine residue allows for Zn(II) regulation in SmtB and, conversely, a glycine results in a lack of regulation by Zn(II) in CadC. We propose that a glycine residue was ancestral whether the repressor binds Zn(II) at a Site 2 like CadC or has no Site 2 like the paralogous ArsR and implies that acquisition of regulatory ability in SmtB was a more recent evolutionary event.

  1. Detection of the secondary, low-affinity β1-adrenoceptor site in living cells using the fluorescent CGP 12177 derivative BODIPY-TMR-CGP

    PubMed Central

    Gherbi, K; Briddon, S J; Hill, S J

    2014-01-01

    Background and Purpose CGP 12177 not only inhibits agonist effects mediated through the catecholamine site of the β1-adrenoceptor with high affinity, but also exhibits agonist effects of its own at higher concentrations through a secondary, low-affinity β1-adrenoceptor site or conformation. β-blocker affinities for this ‘CGP 12177’ site of the human β1-adrenoceptor have thus far only been characterized in functional studies. Here, we used the fluorescent CGP 12177 analogue BODIPY-TMR-CGP to directly investigate receptor–ligand interactions at the secondary binding site of the β1-adrenoceptor. Experimental Approach The human β1-adrenoceptor was stably expressed in CHO cells containing a cAMP response element (CRE)-secreted placental alkaline phosphatase (SPAP) reporter gene construct. Functional responses of BODIPY-TMR-CGP were determined in the CRE-SPAP reporter gene assay, and manual and automated confocal microscopy platforms used to investigate the binding properties of BODIPY-TMR-CGP. Key Results BODIPY-TMR-CGP displayed a pharmacological profile similar to that of CGP 12177, retaining agonist activity at the secondary β1-adrenoceptor site. In confocal microscopy studies, specific BODIPY-TMR-CGP binding allowed clear visualization of β1-adrenoceptors in live cells. Using a wider concentration range of labelled ligand in a high-content fluorescence-based binding assay than is possible in radioligand binding assays, two-site inhibition binding curves of β-adrenoceptor antagonists were revealed in CHO cells expressing the human β1-adrenoceptor, but not the β2-adrenoceptor. Conclusions and Implications The fluorescent CGP 12177 analogue allowed the detection of the β1-adrenoceptor secondary site in both functional and binding studies. This suggests that BODIPY-TMR-CGP presents an important and novel fluorescent tool to investigate the nature of the secondary β1-adrenoceptor site. PMID:25052258

  2. Synthesis and spectroscopic studies on the Schiff base ligand derived from condensation of 2-furaldehyde and 3,3'-diaminobenzidene, L and its complexes with Co(II), Ni(II), Cu(II) and Zn(II): Comparative DNA binding studies of L and its Cu(II) and Zn(II) complexes

    NASA Astrophysics Data System (ADS)

    Shakir, Mohammad; Abbasi, Ambreen; Khan, Asad U.; Khan, Shahper N.

    2011-01-01

    The Schiff base ligand, N,N'-bis-(2-furancarboxaldimine)-3,3'-diaminobenzidene (L) obtained by condensation of 2-furaldehyde and 3,3'-diaminobenzidene, was used to synthesize the mononuclear complexes of the type, [M(L)](NO 3) 2 [M = Co(II), Ni(II), Cu(II) and Zn(II)]. The newly synthesized ligand, (L) and its complexes have been characterized on the basis of the results of the elemental analysis, molar conductance, magnetic susceptibility measurements and spectroscopic studies viz, FT-IR, 1H and 13C NMR, mass, UV-vis and EPR. EPR, UV-vis and magnetic moment data revealed a square planar geometry for the complexes with distortion in Cu(II) complex and conductivity data show a 1:2 electrolytic nature of the complexes. Absorption and fluorescence spectroscopic studies support that Schiff base ligand, L and its Cu(II) and Zn(II) complex exhibit significant binding to calf thymus DNA. The highest binding affinity in case of L may be due to the more open structure as compared to the metal coordinated complexes.

  3. Development of new peptide-based receptor of fluorescent probe with femtomolar affinity for Cu(+) and detection of Cu(+) in Golgi apparatus.

    PubMed

    Jung, Kwan Ho; Oh, Eun-Taex; Park, Heon Joo; Lee, Keun-Hyeung

    2016-11-15

    Developing fluorescent probes for monitoring intracellular Cu(+) is important for human health and disease, whereas a few types of their receptors showing a limited range of binding affinities for Cu(+) have been reported. In the present study, we first report a novel peptide receptor of a fluorescent probe for the detection of Cu(+). Dansyl-labeled tripeptide probe (Dns-LLC) formed a 1:1 complex with Cu(+) and showed a turn-on fluorescent response to Cu(+) in aqueous buffered solutions. The dissociation constant of Dns-LLC for Cu(+) was determined to be 12 fM, showing that Dns-LLC had more potent binding affinity for Cu(+) than those of previously reported chemical probes for Cu(+). The binding mode study showed that the thiol group of the peptide receptor plays a critical role in potent binding with Cu(+) and the sulfonamide and amide groups of the probe might cooperate to form a complex with Cu(+). Dns-LLC detected Cu(+) selectively by a turn-on response among various biologically relevant metal ions, including Cu(2+) and Zn(2+). The selectivity of the peptide-based probe for Cu(+) was strongly dependent on the position of the cysteine residue in the peptide receptor part. The fluorescent peptide-based probe penetrated the living RKO cells and successfully detected Cu(+) in the Golgi apparatus in live cells by a turn-on response. Given the growing interest in imaging Cu(+) in live cells, a novel peptide receptor of Cu(+) will offer the potential for developing a variety of fluorescent probes for Cu(+) in the field of copper biochemistry. PMID:27208475

  4. Synthesis, structures, molecular docking, cytotoxicity and bioimaging studies of two novel Zn(II) complexes.

    PubMed

    Gao, Enjun; Sun, Na; Zhang, Shaozhong; Ding, Yuqing; Qiu, Xue; Zhan, Yang; Zhu, Mingchang

    2016-10-01

    Two novel compounds [Zn2(Endc)2(bipy)2(H2O)3]·4(H2O)·2(O)(1), [Zn2(Endc)2(phen)2(H2O)]·(O)(2) (bipy = 2,2-bipyridine, phen = 1,10-phenanthroline, and Endc = endo-norbornene-cis-5,6-dicarboxylicacid) have been synthesized and characterized. In this paper abbreviations are FS-DNA (fish sperm DNA), HeLa (human cervix epithelia carcinoma cells), KB (human oral epithelial carcinoma cells), LO2 (human liver cell L-O2), EtBr (ethidium bromide), DMF (Dimethyl Formamide), MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium]). The binding of complexes with Fish Sperm DNA were measured by electronic absorption spectra and fluorescence spectroscopy. The ability of these complexes to cleave the pBR322 plasmid DNA or the KB and HeLa DNA extracted in our laboratory was demonstrated by gel electrophoresis assay. The cytotoxic effects of these complexes were examined on two tumor cell lines, HeLa, KBr and one normal cell line LO-2. UV absorption and fluorescence spectra indicate the ability of the complexes bond to DNA with different binding affinity. Gel electrophoresis assay demonstrates which one complex more effective DNA-cleavage activity. The cytotoxic activity of the complexes was tested against two different cancer and one normal cell lines. The two complexes exhibited cytotoxic specificity and significant cancer cell inhibitory rate and lower cytotoxicity toward the normal cell lines. The unique interaction mode with DNA and cancer cells inhibition effect clearly revealed the relationship between the structure and the activity of the novel antitumor agent Zn(II) complexes. PMID:27214507

  5. Determination of aflatoxins and ochratoxin A in high-sugar-content traditional Turkish foods by affinity column cleanup and LC fluorescence detection.

    PubMed

    Senyuva, Hamide Z; Cimen, Dilek; Gilbert, John

    2009-01-01

    The effectiveness of an affinity column cleanup procedure followed by LC with fluorescence detection was established for the determination of aflatoxins and ochratoxin A in high-sugar-content traditional Turkish foods. Traditional foods, such as baklava (finely layered pastry filled with nuts and steeped in syrup), halvah (containing sesame paste and pistachios), cevizli sucuk (a confection made of grape juice boiled and dried on strings of nuts), Turkish delight (containing hazelnuts, pistachios, or walnuts), and pişmaniye (candy made of sugar, butter, and flour), were tested, and the performance of the method was established with spiked samples. To examine the robustness of the methodology, baklava was prepared from raw materials and spiked at the initial stage of dry ingredients and through subsequent stages of preparation of dough, after cooking, and after addition of syrup and nuts. For all products, the analytical method required grinding the composite foodstuff under liquid nitrogen to form a fine powder, which was then thoroughly mixed before subsampling. After vortex extraction into methanol-water (aflatoxins) and aqueous sodium bicarbonate (ochratoxin A), the sample was filtered, diluted with phosphate-buffered saline, and then passed through either an aflatoxin or ochratoxin A affinity column before HPLC analysis with fluorescence detection (using post-column bromination for the aflatoxins). In all the traditional Turkish products, the recovery of aflatoxin B1 ranged from 77 to 98%, and LODs were <0.1 microg/kg. For ochratoxin A, the recoveries were from 88 to 93% and LODs were similarly <0.1 microLg/kg. Despite the complex nature of these traditional Turkish foods, which frequently contain products from sugar caramelization, there was no evidence of any interfering co-extractives, and the method has proved to be robust enough to be used for food control purposes. PMID:19714981

  6. Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

    PubMed Central

    Grate, Jay W.; Tyler, Abby; Ozanich, Richard M.; Miller, Keith D.; Lou, Jianlong; Marks, James D.; Bruckner-Lea, Cynthia J.

    2009-01-01

    A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum neurotoxin serotype A (BoNT/A) using a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. Detection to 31 pM with a total incubation time of 3 hours was demonstrated. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. The beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell equipped with a fiber optic system for fluorescence measurements. In PBS buffer, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach. PMID:19643593

  7. Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

    SciTech Connect

    Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.; Ozanich, Richard M.; Miller, Keith D.; Lou, Jianlong; Marks, James D.; Bruckner-Lea, Cindy J.

    2009-09-01

    A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment was demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.

  8. Metal-enhanced fluorescence-based core-shell Ag@SiO₂ nanoflares for affinity biosensing via target-induced structure switching of aptamer.

    PubMed

    Lu, Lu; Qian, Yunxia; Wang, Lihui; Ma, Keke; Zhang, Yaodong

    2014-02-12

    One of the great challenges in metal-enhanced fluorescence (MEF) technology is the achievement of distance modulation with nanometer accuracy between the fluorophore and metal surface to obtain maximum enhancement. We propose an MEF-based core-shell Ag@SiO2 nanoflare for distance control via the thickness of silica shell with cooperation of DNA hybridization. The nanoflare contains a 50 nm spherical silver nanoparticle (Ag NP) core, a 8 nm silica shell, and cyanine (Cy5)-labeled aptamer hybridized with a complementary DNA (cDNA) immobilized onto the shell surface. The formation of the Cy5-labeled aptamer/cDNA duplex on the Ag@SiO2 NP surface results in the confinement of Cy5 to the shell surface and an increase in the fluorescence of Cy5 with a 32-fold enhancement factor in bulk solution (signal-on). In the presence of affinity-binding targets, the Cy5-labeled aptamers confined onto the Ag@SiO2 NP surface dissociate from their cDNA into the solution because of structure switching. The target-induced release of aptamer leads to a reduction in the enhanced fluorescence signal of the labeled Cy5 moiety (signal-off). Thus, the nanoflare can be used as a sensor for target recognition. Using adenosine-5'-triphosphate (ATP) aptamer, detection of ATP has a linear response from 0 to 0.5 mM and a detection limit of 8 μM. With various types of DNA probes immobilized onto the core-shell Ag@SiO2 NPs, the MEF-based nanoflare has provided an effective platform for the detection and quantification of a broad range of analytes, such as mRNA regulation and detection, cell sorting, and gene profiling. PMID:24480015

  9. Estimation of interaction between oriented immobilized green fluorescent protein and its antibody by high performance affinity chromatography and molecular docking.

    PubMed

    Li, Qian; Wang, Jing; Yang, Lingjian; Gao, Xiaokang; Chen, Hongwei; Zhao, Xinfeng; Bian, Liujiao; Zheng, Xiaohui

    2015-07-01

    Although green fluorescence protein (GFP) and its antibody are widely used to track a protein or a cell in life sciences, the binding behavior between them remains unclear. In this work, diazo coupling method that synthesized a new stationary GFP was oriented immobilized on the surface of macro-porous silica gel by a phase. The stationary phase was utilized to confirm the validation of injection amount-dependent analysis in exploring protein-protein interaction that use GFP antibody as a probe. GFP antibody was proved to have one type of binding site on immobilized GFP. The number of binding site and association constant were calculated to be (6.41 ± 0.76) × 10(-10) M and (1.39 ± 0.12) × 10(9) M(-1). Further analysis by molecular docking showed that the binding of GFP to its antibody is mainly driven by hydrogen bonds and salt bridges. These results indicated that injection amount-dependent analysis is capable of exploring the protein-protein interactions with the advantages of ligand and time saving. It is a valuable methodology for the ligands, which are expensive or difficult to obtain. PMID:25727342

  10. Revised stability constant, spectroscopic properties and binding mode of Zn(II) to FluoZin-3, the most common zinc probe in life sciences.

    PubMed

    Marszałek, I; Krężel, A; Goch, W; Zhukov, I; Paczkowska, I; Bal, W

    2016-08-01

    2-[2-[2-[2-[bis(carboxylatomethyl)amino]-5-methoxyphenoxy]ethoxy]-4-(2,7-difluoro-3-oxido-6-oxo-4a,9a-dihydroxanthen-9-yl)anilino]acetate (FluoZin-3) is used very broadly in life sciences as intra- and extracellular Zn(II) sensor selective for Zn(II) over Co(II), Ca(II) and Mg(II) ions at their physiological concentrations. It has been used for determination of relative and absolute levels of exchangeable Zn(II) in cells and extracellular fluids. Despite its popularity, the knowledge of its acid/base and Zn(II) coordination abilities and of its spectroscopic properties remained very limited. Also the published conditional dissociation constant ((C)Kd) values at pH7.4 are slightly discrepant, (15nM or 8.9nM). In this work we determined the (C)Kd for Zn(II) complexation by FluoZin-3 at pH7.4 with nitrilotriacetic acid (NTA) as competitor using two independent methods: fluorimetry and UV-Vis spectroscopy. For the first time, we investigated FluoZin-3 alone and complexed with Zn(II) in the wide range of pH, determining the total of eight pKa values from fluorescence spectra and from various regions of UV-Vis spectra. The validated values of (C)Kd (9.1±0.4nM; -log (C)Kd=8.04) and of the absolute (pH-independent) stability constant log βZnL (8.16±0.05) were provided by fluorescence spectroscopy experiments performed at 1μM concentrations. Our experiments demonstrated that both of aminocarboxylate moieties of FluoZin-3 bind the Zn(II) ion synergistically. PMID:27216451

  11. In-line coupling of an aptamer based miniaturized monolithic affinity preconcentration unit with capillary electrophoresis and Laser Induced Fluorescence detection.

    PubMed

    Marechal, A; Jarrosson, F; Randon, J; Dugas, V; Demesmay, C

    2015-08-01

    A composite 30-cm capillary was prepared. The head of the capillary was a 1.5-cm original and miniaturized aptamer-based monolithic affinity support that was in-line coupled to the end of the capillary used for capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The device was used for the preconcentration, separation and quantification of ochratoxin A (OTA) as a test solute. The 1.5-cm preconcentration unit consists of a fritless affinity monolithic bonded with 5'-SH-modified oligonucleotide aptamers. A vinyl spacer was used for thiol-ene photoclick chemistry with a 5min irradiation at 365nm. Photografting allowed to confine the binding reaction to the desired silica monolithic segment, upstream the empty section of the CE capillary using an UV mask. The photografting procedure was optimized preparing 10-cm capillary monoliths for nano-LC. The retention factors of cationic solutes in ion-exchange nano-LC allowed to follow the aptamer binding on the monolith. The reproducibility of the photografting process was satisfactory with inter-capillary variation lower than 10%. The aptamer bonding density can be increased by successive graftings of 100μM aptamer concentration solution (5pmol/cm/grafting). The optimal conditions to successfully perform the in-line coupling (preconcentration, elution and separation of OTA) with the composite capillary were adjusted depending on individual requirements of each step but also insuring compatibility. Under optimized conditions, OTA was successfully preconcentrated and quantified down to 0.1pg (percolation of 2.65μL of a 40ng/L OTA solution). A quantitative recovery of OTA (93±2%) was achieved in a single elution of 30pg percolated OTA amount. The reproducibility of the overall process was satisfactory with a relative standard deviation lower than 10% with negligible non-specific adsorption. This device was applied for the preconcentration and analysis of OTA in beer and wine at the ppb level within

  12. Rational synthesis of an exceptionally stable Zn(II) metal-organic framework for the highly selective and sensitive detection of picric acid.

    PubMed

    Hu, Yingli; Ding, Meili; Liu, Xiao-Qin; Sun, Lin-Bing; Jiang, Hai-Long

    2016-04-28

    Based on an organic ligand involving both carboxylate and tetrazole groups, a chemically stable Zn(II) metal-organic framework has been rationally synthesized and behaves as a fluorescence chemosensor for the highly selective and sensitive detection of picric acid, an extremely hazardous and strong explosive. PMID:27046028

  13. Hydrogen bond-assisted aggregation-induced emission and application in the detection of the Zn(ii) ion.

    PubMed

    Wang, Dan; Li, Shu-Mu; Li, Yu-Fei; Zheng, Xiang-Jun; Jin, Lin-Pei

    2016-05-28

    The compounds of 3-aminopyridine-2-carboxylic acid with K(+) (1) and Zn(2+) (2) were found to be AIE-active. The AIE behaviours could be attributed to the restriction of intramolecular rotation (RIR) and vibration (RIV) via hydrogen bonds, resulting in rigidity enhancement of the molecules. An AIE-based fluorescence turn on chemosensor for the Zn(ii) ion has been developed in aqueous media with high selectivity and sensitivity. PMID:27126357

  14. Enhancing the Photostability of Arylvinylenebipyridyl Compounds as Fluorescent Indicators for Intracellular Zinc(II) Ions.

    PubMed

    Yuan, Zhao; Younes, Ali H; Allen, John R; Davidson, Michael W; Zhu, Lei

    2015-06-01

    Arylvinylenebipyridyl (AVB) ligands are bright, zinc(II)-sensitive fluoroionophores. The applicability of AVBs as fluorescent indicators for imaging cellular zinc(II), however, is limited by low photostability, partially attributable to the photoisomerization of the vinylene functionality. Two configurationally immobilized (i.e., "locked") AVB analogues are prepared in this work. The zinc(II)-sensitive photophysical properties and zinc(II) affinities of both AVBs and their locked analogues are characterized in organic and aqueous media. The zinc(II) sensitivity of the emission is attributed to the zinc(II)-dependent energies of the charge transfer excited states of these compounds. The configurationally locked ligands have improved photostability, while maintaining the brightness and zinc(II) sensibility of their AVB progenitors. The feasibility of the "locked" AVB analogues with improved photostability for imaging intracellular Zn(II) of eukaryotic cells using laser confocal fluorescence microscopy is demonstrated. PMID:25942357

  15. Enhancing the Photostability of Arylvinylenebipyridyl Compounds as Fluorescent Indicators for Intracellular Zinc(II) Ions

    PubMed Central

    Yuan, Zhao; Younes, Ali H.; Allen, John R.; Davidson, Michael W.; Zhu, Lei

    2015-01-01

    Arylvinylenebipyridyl (AVB) ligands are bright, zinc(II)-sensitive fluoroionophores. The applicability of AVBs as fluorescent indicators for imaging cellular zinc(II), however, is limited by low photostability, partially attributable to the photoisomerization of the vinylene functionality. Two configurationally immobilized (i.e., “locked”) AVB analogues are prepared in this work. The zinc(II)-sensitive photophysical properties and zinc(II) affinities of both AVBs and their locked analogues are characterized in organic and aqueous media. The zinc(II) sensitivity of the emission is attributed to the zinc(II)-dependent energies of the charge transfer excited states of these compounds. The configurationally locked ligands have improved photostability, while maintaining the brightness and zinc(II) sensibility of their AVB progenitors. The feasibility of the “locked” AVB analogues with improved photostability for imaging intracellular Zn(II) of eukaryotic cells using laser confocal fluorescence microscopy is demonstrated. PMID:25942357

  16. Rapid screening method for quinolone residues in livestock and fishery products using immobilised metal chelate affinity chromatographic clean-up and liquid chromatography-fluorescence detection.

    PubMed

    Takeda, N; Gotoh, M; Matsuoka, T

    2011-09-01

    An efficient LC method was developed for screening the presence of quinolones (QLs)--comprising fluoroquinolones (FQs) and acidic quinolones (AQs)--residues in various livestock and fishery products. Targeted analytes were for nine FQs of marbofloxacin (MAR), ofloxacin (OFL), norfloxacin (NOR), ciprofloxacin (CIP), enrofloxacin (ENR), danofloxacin (DAN), orbifloxacin (ORB), difloxacin (DIF) and sarafloxacin (SAR), and three AQs of oxolinic acid (OXA), nalidixic acid (NAL) and flumequine (FMQ). Samples comprised ten different food products covering five matrices: muscle (cattle, swine and chicken), liver (chicken), raw fish (shrimp and salmon), egg (chicken), and processed food (ham, sausage and fish sausage). This method involved a simple extraction with (1:1) acetonitrile-methanol, a highly selective clean-up with an immobilised metal chelate affinity column charged with Fe(3+), a fast isocratic LC analysis using a short column (20 mm × 4.6 mm, 3 µm) with a mobile phase of (15:85:0.1) methanol/water/formic acid, and fluorescence detection (excitation/emission wavelengths of 295 nm/455 nm for FQs (495 nm for MAR), and 320 nm/365 nm for AQs). Among FQs, pairs of NOR/OFL, ORB/DIF and ENR/DAN were incompletely resolved. A confirmatory LC run with a Mg(2+) containing methanolic mobile phase was also proposed for the samples suspected of being positive. The optimised method gave satisfactory recoveries of 88.5% (56.1-108.6%) and 78.7% (44.1-99.5%) for intra- and inter-day assays with relative standard deviations of 7.2% (0.7-18.4%) and 6.8% (1.4-16.6%), respectively. Limits of quantitation ranged from 0.8 µg kg(-1) (DAN) to 6.5 µg kg(-1) (SAR). This method was successfully employed to analyse 113 real samples and two positive samples were found: fish sausage (CIP 990 µg kg(-1)) and shrimp (ENR 20 µg kg(-1)). PMID:21749230

  17. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

    Presents selected recent advances in immobilization chemistry which have important connections to affinity chromatography. Discusses ligand immobilization and support modification. Cites 51 references. (CS)

  18. Synthesis, spectroscopic, photoluminescence properties and biological evaluation of novel Zn(II) and Al(III) complexes of NOON tetradentate Schiff bases

    NASA Astrophysics Data System (ADS)

    Abdel Aziz, Ayman A.; Badr, Ibrahim H. A.; El-Sayed, Ibrahim S. A.

    2012-11-01

    Novel mononuclear Zn(II) and Al(III) complexes were synthesized from the reactions of Zn(OAc)2·2H2O and anhydrous AlCl3 with neutral N2O2 donor tetradentate Schiff bases; N,N'bis(salicylaldehyde)4,5-dimethyl-1,2-phenylenediamine (H2L1) and N,N'bis(salicylaldehyde)4,5-dichloro-1,2-phenylenediamine (H2L2). The new complexes were fully characterized by using micro analyses (CHN), FT-IR, 1H NMR, UV-Vis spectra and thermal analysis. The analytical data have been showed that, the stoichiometry of the complexes is 1:1. Spectroscopic data suggested tetrahedral and square pyramidal geometries for Zn(II) and Al(III) complexes, respectively. The synthesized Zn(II), and Al(III) complexes exhibited intense fluorescence emission in the visible region upon UV-excitation in methylene chloride solution at ambient temperature. This high fluorescence emission was assigned to the strong coordination of the ligands to the small and the highly charged Zn(II) and Al(III) ions. Such strong coordination seems to extend the π-conjugation of the complexes. Thermal analysis measurements indicated that the complexes have good thermal stability. As a potential application the biological activity (e.g., antimicrobial action) of the prepared ligands and complexes was assessed by in-vitro testing of their effect on the growth of various strains of bacteria and fungi.

  19. Small-Molecule Fluorescent Sensors for Investigating Zinc Metalloneurochemistry

    PubMed Central

    Nolan, Elizabeth M.; Lippard, Stephen J.

    2008-01-01

    Conspectus Metal ions are involved in many neurobiological processes relevant to human health and disease. The metalloneurochemistry of Zn(II) is of substantial current interest. Zinc is the second most abundant d-block metal ion in the human brain and its distribution varies, with relatively high concentrations found in the hippocampus. Brain zinc is generally divided into two categories: protein-bound and loosely-bound. The latter pool is also referred to as histochemically observable, chelatable, labile, or mobile zinc. The neurophysiological and neuropathological significance of such mobile Zn(II) remains enigmatic. Studies of Zn(II) distribution, translocation, and function in vivo require tools for its detection. Because Zn(II) has a closed-shell d10 configuration and no convenient spectroscopic signature, fluorescence is a suitable method for monitoring Zn(II) in biological contexts. This Account summarizes work by our laboratory addressing the design, preparation, characterization, and use of small-molecule fluorescent sensors for imaging mobile Zn(II) in living cells and samples of brain tissue. These sensors provide “turn-on” or ratiometric Zn(II) detection in aqueous solution at neutral pH. By making alterations to the Zn(II)-binding unit and fluorophore platform, we have devised sensors with varied photophysical and metal-binding properties. We used several of these probes to image Zn(II) distribution, uptake, and mobilization in a variety of cell types, including neuronal cultures. Goals for the future include developing strategies for multi-color imaging, further defining the quenching and turn-on mechanisms of the sensors, and employing the probes to elucidate the functional significance of Zn(II) in neurobiology. PMID:18989940

  20. Synthesis, crystal structures, biological activities and fluorescence studies of transition metal complexes with 3-carbaldehyde chromone thiosemicarbazone.

    PubMed

    Li, Yong; Yang, Zheng-Yin; Wu, Jin-Cai

    2010-12-01

    3-carbaldehyde chromone thiosemicarbazone (L) and its transition metal complexes were synthesized and characterized systematically. Crystal structures of Zn(II) and Ni(II) complexes were determined by single crystal X-ray diffraction analysis. Zn(II) complex exhibits blue fluorescence under UV light and its fluorescent property in solid state was investigated. Interactions of ligand and Cu(II), Zn(II) and Ni(II) complexes with DNA were investigated by spectral and viscosity studies, indicating the compounds bind to DNA via intercalation and Zn(II) complex binds to DNA most strongly. Antioxidant tests in vitro show the compounds possess significant antioxidant activity against superoxide and hydroxyl radicals, and the scavenging effects of Cu(II) complex are stronger than Zn(II), Ni(II) complexes and some standard antioxidants, such as mannitol and vitamin C. PMID:20884087

  1. Design, Synthesis, and Initial Evaluation of Affinity-Based Small-Molecule Probes for Fluorescent Visualization and Specific Detection of Keap1.

    PubMed

    Lu, Mengchen; Zhou, Hai-Shan; You, Qi-Dong; Jiang, Zhengyu

    2016-08-11

    Keap1 is a pluripotent protein which plays a predominant role in cellular homeostasis and stress responses. Given that the cellular environment is quite dynamic and versatile, further investigation of the function of Keap1 depends on tools for specific and real-time detection of Keap1. Herein, we report the development of functional affinity-based small-molecule probes which can overcome some shortcomings of current methods and be applied in further studying the function of Keap1. PMID:27409246

  2. Synthesis, characterization, crystal structure, DNA- and HSA-binding studies of a dinuclear Schiff base Zn(II) complex derived from 2-hydroxynaphtaldehyde and 2-picolylamine

    NASA Astrophysics Data System (ADS)

    Kazemi, Zahra; Rudbari, Hadi Amiri; Mirkhani, Valiollah; Sahihi, Mehdi; Moghadam, Majid; Tangestaninejad, Sharam; Mohammadpoor-Baltork, Iraj

    2015-09-01

    A tridentate Schiff base ligand NNO donor (HL: 1-((E)-((pyridin-2-yl)methylimino)methyl)naphthalen-2-ol was synthesized from condensation of 2-hydroxynaphtaldehyde and 2-picolylamine. Zinc complex, Zn2L2(NO3)2, was prepared from reaction of Zn(NO3)2 and HL at ambient temperature. The ligand and complex were characterized by FT-IR, 1H NMR, 13C NMR and elemental analysis (CHN). Furthermore, the structure of dinuclear Zn(II) complex was determined by single crystal X-ray analysis. The complex, Zn2L2(NO3)2, is centrosymmetric dimer in which deprotonated phenolates bridge the two Zn(II) atoms and link the two halves of the dimer. In the structure, Zinc(II) ions have a highly distorted six-coordinate structure bonded to two oxygen atoms from a bidentate nitrate group, the pyridine nitrogen, an amine nitrogen and phenolate oxygens. The interaction of dinuclear Zn(II) complex with fish sperm DNA (FS-DNA) and HSA was investigated under physiological conditions using fluorescence quenching, UV-Vis spectroscopy, molecular dynamics simulation and molecular docking methods. The estimated binding constants for the DNA-complex and HSA-complex were (3.60 ± 0.18) × 104 M-1 and (1.35 ± 0.24) × 104 M-1, respectively. The distance between dinuclear Zn(II) complex and HSA was obtained based on the Förster's theory of non-radiative energy transfer. Molecular docking studies revealed the binding of dinuclear Zn(II) complex to the major groove of FS-DNA and IIA site of protein by formation of hydrogen bond, π-cation and hydrophobic interactions.

  3. Fluorescent detection of apoptotic cells using a family of zinc coordination complexes with selective affinity for membrane surfaces that are enriched with phosphatidylserine.

    SciTech Connect

    Smith, Bradley D.; Lambert, Timothy N.; Lakshmi, C.; Hanshaw, Roger, G.

    2005-03-01

    The appearance of phosphatidylserine on the membrane surface of apoptotic cells (Jurkat, CHO, HeLa) is monitored by using a family of bis(Zn{sup 2+}-2,2{prime}-dipicolylamine) coordination compounds with appended fluorescein or biotin groups as reporter elements. The phosphatidylserine affinity group is also conjugated directly to a CdSe/CdS quantum dot to produce a probe suitable for prolonged observation without photobleaching. Apoptosis can be detected under a wide variety of conditions, including variations in temperature, incubation time, and binding media. Binding of each probe appears to be restricted to the cell membrane exterior, because no staining of organelles or internal membranes is observed.

  4. Selective inhibition of the hypoxia-inducible factor prolyl hydroxylase PHD3 by Zn(II).

    PubMed

    Na, Yu-Ran; Woo, Dustin J; Choo, Hyunah; Chung, Hak Suk; Yang, Eun Gyeong

    2015-07-01

    We report herein that Zn(II) selectively inhibits the hypoxia-inducible factor prolyl hydroxylase PHD3 over PHD2, and does not compete with Fe(II). Independent of the oligomer formation induced by Zn(II), inhibition of the activity of PHD3 by Zn(II) involves Cys42 and Cys52 residues distantly located from the active site. PMID:26051901

  5. Flash-induced blocking of the high-affinity manganese-binding site in photosystem II by iron cations: dependence on the dark interval between flashes and binary oscillations of fluorescence yield.

    PubMed

    Semin, Boris K; Seibert, Michael

    2006-12-21

    Incubation of Fe(II) cations with Mn-depleted PSII membranes (PSII(-Mn)) under weak continuous light is accompanied by blocking of the high-affinity, Mn-binding (HAZ) site with ferric cations (Semin, B.K. et al. Biochemistry 2002, 41, 5854-5864). In this study we investigated the blocking yield under single-turnover flash conditions. The flash-probe fluorescence method was used to estimate the blocking efficiency. We found that the yield of blocking increases with flash number and reaches 50% after 7 flashes. When the dark interval between the flashes (Delta t) was varied, we found that the percentage of blocking decreases at Delta t < 100 ms (t 1/2, 4-10 ms). No inhibition of the blocking yield was found at longer time intervals (as with photoactivation). This result shows the necessity of a dark rearrangement during the blocking process (the dual-site hypothesis described in the text) and indicates the formation of a binuclear iron center. During the blocking experiments, we found a binary oscillation of the Fmax elicited during a train of flashes. The oscillations were observed only in the presence of Fe(II) cations or other electron donors (including Mn(II)) but not in the presence of Ca2+. Chelators had no effect on the oscillations. Our results indicate that the oscillations are due to processes on the acceptor side of PSII and to the appearance of "acceptor X" after odd flashes. Acceptor X is reduced by QA- at very high rate (<2 ms), is not sensitive to DCMU, and is rather stable in the dark (t l/2 approximately 2 min). These properties are similar to those of nonheme Fe(III) (Fe(III)NHI). When Fe(II)NHI was oxidized with ferricyanide (Fe(CN)6), the fluorescence decay kinetics and yield of fluorescence were identical to those observed when the sample was exposed to 1 flash prior to the fluorescence measurement. We suggest that acceptor X is Fe(III)NHI, oxidized by the semiquinone form of QB-. This is similar to the mechanism of "reduction-induced oxidation

  6. Measuring an antibody affinity distribution molecule by molecule

    SciTech Connect

    Bradbury, Andrew M; Werner, James H; Temirov, Jamshid

    2008-01-01

    Single molecule fluorescence mIcroscopy was used to observe the binding and unbinding of hapten decorated quantum dots with individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.

  7. A convenient fluorescent method to simultaneously determine the enantiomeric composition and concentration of functional chiral amines.

    PubMed

    Huang, Zeng; Yu, Shanshan; Zhao, Xue; Wen, Kaili; Xu, Yimang; Yu, Xiaoqi; Xu, Yong; Pu, Lin

    2014-12-01

    A 1,1'-bi-2-naphthol (BINOL)-based chiral aldehyde in combination with Zn(II) shows a highly enantioselective fluorescent response toward functional chiral amines at λ>500 nm. However, the combination of salicylaldehyde and Zn(II) gives the same fluorescent enhancement for both enantiomers of a functional chiral amine at λ=447 nm. By using the fluorescent responses of the combination of the BINOL-based chiral aldehyde, salicylaldehyde and Zn(II) at the two emission wavelengths, both the concentration and enantiomeric composition of functional chiral amines such as amino alcohols, diamines, and amino acids can be simultaneously determined by a single fluorescent measurement. This work provides a simple and convenient method for chiral assay. PMID:25348091

  8. Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Walters, Rodney R.

    1985-01-01

    Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)

  9. A Biphenol-Based Chemosensor for Zn(II) and Cd(II) Metal Ions: Synthesis, Potentiometric Studies, and Crystal Structures.

    PubMed

    Ambrosi, Gianluca; Formica, Mauro; Fusi, Vieri; Giorgi, Luca; Macedi, Eleonora; Micheloni, Mauro; Paoli, Paola; Rossi, Patrizia

    2016-08-01

    We synthesized and characterized the ligand N,N'-bis[(2,2'-dihydroxybiphen-3-yl)methyl]-N,N'-dimethylethylenediamine (L), which contains two biphenol moieties linked as side arms to an N,N'-dimethylethylenediamine scaffold. The ligand is highly soluble in a 50/50 (v/v) water/ethanol mixture and, in its deprotonated form H-2L(2-), is able to coordinate transition-metal ions such as Ni(II), Zn(II), Cu(II), Cd(II), and Pd(II). The crystal structures of [Ni(H-2L)·2n-BuOH], [Ni(H-2L)·2MeOH], [Cd(H-2L)·2DMF], [Cu(H-2L)(DMF)], and [Pd(H-2L)(DMF)] were also determined and described. Potentiometric titrations were carried out in a mixed solvent with Zn(II), Cu(II), and Ni(II) metal ions to determine the acid-base and stability constants. L was highly fluorescent in the visible range (400 nm). Moreover, its emission intensity increased upon the addition of Zn(II) or Cd(II) ions in an ethanol/water solution and behaved as a chemosensor for the presence of these ions in the solution. PMID:27439670

  10. Synthesis Characterization and DNA Interaction Studies of a New Zn(II) Complex Containing Different Dinitrogen Aromatic Ligands

    PubMed Central

    Shahabadi, Nahid; Mohammadi, Somaye

    2012-01-01

    A mononuclear complex of Zn(II), [Zn(DIP)2 (DMP)] (NO3)2·2H2O in which DIP is 4,7-diphenyl-1,10-phenanthroline and DMP is 4,4′-dimethyl-2,2′-bipyridine has been prepared and characterized by 1HNMR spectroscopy, FT-IR, UV-Vis and elemental analysis techniques. DNA-binding properties of the complex were studied using UV-vis spectra, circular dichroism (CD) spectra, fluorescence, cyclic voltammetry (CV), and viscosity measurements. The results indicate that this zinc(II) complex can intercalate into the stacked base pairs of DNA and compete with the strong intercalator ethidium bromide for the intercalative binding sites. PMID:22956919

  11. Syntheses, structures and properties of Zn(II) and Cu(II) complexes based on N2-2-methylenepyridinyl 1,2,3-triazole ligand

    NASA Astrophysics Data System (ADS)

    Chen, Yunfeng; Wu, Jun; Ma, Shan; Zhou, Shilei; Meng, Xianggao; Jia, Lihui; Pan, Zhiquan

    2015-06-01

    Four new Zn(II) and Cu(II) coordinated polymers ([ZnL2N3]ClO4 (1), [Cu2L2(CH3CN)]Cl4 (2) [CuL](NO3)2 (3), [Cu(H2O)L](SO4) (4) L = 2-((4-phenyl-2H-1,2,3-triazol-2-yl)methyl) pyridine (ptmp)) have been reported. All the compounds have been characterized by IR spectrum, elemental analyses and X-ray crystallography diffraction. Single-crystal X-ray diffraction analyses show that one-dimensional polymers are formed in these four complexes. Chain-like structures are formed in complex 1, 2 and 3, which are connected by azide, chloride and nitrate anions, respectively. In complex 4, one-dimensional left-handed polymer is formed by a μ2-SO4 bridge. The fluorescent and electrochemical properties of these four complexes were investigated. It was found that these three Cu(II) complexes displayed a quenching of fluorescence, while Zn(II) complex exhibited a clear enhanced fluorescence.

  12. Impacts of aqueous Mn(II) on the sorption of Zn(II) by hexagonal birnessite.

    PubMed

    Lefkowitz, Joshua P; Elzinga, Evert J

    2015-04-21

    We used a combination of batch studies and spectroscopic analyses to assess the impacts of aqueous Mn(II) on the solubility and speciation of Zn(II) in anoxic suspensions of hexagonal birnessite at pH 6.5 and 7.5. Introduction of aqueous Mn(II) into pre-equilibrated Zn(II)-birnessite suspensions leads to desorption of Zn(II) at pH 6.5, but enhances Zn(II) sorption at pH 7.5. XAS results show that Zn(II) adsorbs as tetrahedral and octahedral triple-corner-sharing complexes at layer vacancy sites when reacted with birnessite in the absence of Mn(II). Addition of aqueous Mn(II) causes no discernible change in Zn(II) surface speciation at pH 6.5, but triggers conversion of adsorbed Zn(II) into spinel Zn(II)1-xMn(II)xMn(III)2O4 precipitates at pH 7.5. This conversion is driven by electron transfer from adsorbed Mn(II) to structural Mn(IV) generating Mn(III) surface species that coprecipitate with Zn(II) and Mn(II). Our results demonstrate substantial production of these reactive Mn(III) surface species within 30 min of contact of the birnessite substrate with aqueous Mn(II). Their importance as a control on the sorption and redox reactivity of Mn-oxides toward Zn(II) and other trace metal(loid)s in environments undergoing biogeochemical manganese redox cycling requires further study. PMID:25790186

  13. Calcium ion gradients modulate the zinc affinity and antibacterial activity of human calprotectin.

    PubMed

    Brophy, Megan Brunjes; Hayden, Joshua A; Nolan, Elizabeth M

    2012-10-31

    Calprotectin (CP) is an antimicrobial protein produced and released by neutrophils that inhibits the growth of pathogenic microorganisms by sequestering essential metal nutrients in the extracellular space. In this work, spectroscopic and thermodynamic metal-binding studies are presented to delineate the zinc-binding properties of CP. Unique optical absorption and EPR spectroscopic signatures for the interfacial His(3)Asp and His(4) sites of human calprotectin are identified by using Co(II) as a spectroscopic probe. Zinc competition titrations employing chromophoric Zn(II) indicators provide a 2:1 Zn(II):CP stoichiometry, confirm that the His(3)Asp and His(4) sites of CP coordinate Zn(II), and reveal that the Zn(II) affinity of both sites is calcium-dependent. The calcium-insensitive Zn(II) competitor ZP4 affords dissociation constants of K(d1) = 133 ± 58 pM and K(d2) = 185 ± 219 nM for CP in the absence of Ca(II). These values decrease to K(d1) ≤ 10 pM and K(d2) ≤ 240 pM in the presence of excess Ca(II). The K(d1) and K(d2) values are assigned to the His(3)Asp and His(4) sites, respectively. In vitro antibacterial activity assays indicate that the metal-binding sites and Ca(II)-replete conditions are required for CP to inhibit the growth of both Gram-negative and -positive bacteria. Taken together, these data provide a working model whereby calprotectin responds to physiological Ca(II) gradients to become a potent Zn(II) chelator in the extracellular space. PMID:23082970

  14. Ternary complex formation and competition quench fluorescence of ZnAF family zinc sensors.

    PubMed

    Staszewska, Anna; Kurowska, Ewa; Bal, Wojciech

    2013-11-01

    Our current understanding of the intracellular thermodynamics and kinetics of Zn(ii) ions is largely based on the application of fluorescent sensor molecules, used to study and visualize the concentration, distribution and transport of Zn(ii) ions in real time. Such agents are designed for high selectivity for zinc in respect to other biological metal ions. However, the issue of their sensitivity to physiological levels of low molecular weight Zn(ii) ligands (LMWLs) has not been addressed. We followed the effects of eight such compounds on the fluorescence of ZnAF-1 and ZnAF-2F, two representatives of the ZnAF family of fluorescein-based zinc sensors containing the N,N-bis(2-pyridylmethyl)ethylenediamine chelating unit. Fluorescence titrations of equimolar Zn(ii)-ZnAF-1 and Zn(ii)-ZnAF-2F solutions with acetate, phosphate, citrate, glycine, glutamic acid, histidine, ATP and GSH demonstrated strong fluorescence quenching. These results are interpreted in terms of an interplay of the formation of the [ZnAF-Zn(ii)-LMWL] ternary complexes and the competition for Zn(ii) between ZnAF and LMWLs. UV-vis spectroscopic titrations revealed the existence of supramolecular interactions between the fluorescein moiety of ZnAF-1 and ATP and His, which, however, did not contribute to fluorescence quenching. Therefore, the obtained results show that the ZnAF sensors, other currently used zinc sensors containing the N,N-bis(2-pyridylmethyl)ethylenediamine unit, and, in general, all sensors that do not saturate the Zn(ii) coordination sphere may co-report cellular metabolites and Zn(ii) ions, leading to misrepresentations of the concentrations and fluxes of biological zinc. PMID:23939683

  15. Adsorption and Precipitation of Aqueous Zn(II) on Hematite Nano- and Microparticles

    SciTech Connect

    Ha, Juyoung; Farges, Francois; Brown, Gordon E. Jr.

    2007-02-02

    As part of a study of the effect of particle size on reactivity of hematite to aqueous metal ions, the sorption of Zn(II) on hematite nanoparticles and microparticles was examined over a wide range of Zn(II) concentrations using Zn K-edge EXAFS. When reacted with nanoparticles at pH 5.5 and low Zn(II) sorption densities (0.04 {<=} {gamma} < 2.76 {mu}mol/m2), Zn(II) formed five-coordinated or a mixture of four- and six-coordinated surface complexes with an average Zn-O distance of 2.04({+-}0.02)A. At pH 5.5 and high Zn(II) sorption densities (2.76 {<=} {gamma} {<=} 3.70 {mu}mol/m2), formation of surface precipitates is suggested based on the presence of second-shell Zn and multiple scattering features in the Fourier transform (FT) of the EXAFS spectra. EXAFS fitting of these high {gamma} samples yielded an average first-shell Zn-O distance of 2.10({+-}0.02)A, with second-shell Zn-Fe and Zn-Zn distances of 3.23({+-}0.03)A and 3.31({+-}0.03)A, respectively. Qualitative comparison between the EXAFS spectra of these sorption samples and that of amorphous zinc hydroxide and Zn-bearing hydrotalcite indicates the development of surface precipitates with increasing {gamma}. EXAFS spectra of Zn(II) sorbed on hematite microparticles under similar experimental conditions showed no evidence for surface precipitates even at the highest Zn surface coverage ({gamma} = 4 {mu}mol/m2). These results indicate that reactivities of hematite nanoparticles and macroparticles differ with respect to Zn(II)aq, depending on Zn(II) sorption density. We suggest that the degree of hematite crystallinity affects the reactivity of hematite surfaces toward Zn(II)aq and the formation of the Zn(II) surface complexes.

  16. Zn(II) and Hg(II) binding to a designed peptide that accommodates different coordination geometries.

    PubMed

    Szunyogh, Dániel; Gyurcsik, Béla; Larsen, Flemming H; Stachura, Monika; Thulstrup, Peter W; Hemmingsen, Lars; Jancsó, Attila

    2015-07-28

    Designed metal ion binding peptides offer a variety of applications in both basic science as model systems of more complex metalloproteins, and in biotechnology, e.g. in bioremediation of toxic metal ions, biomining or as artificial enzymes. In this work a peptide (HS: Ac-SCHGDQGSDCSI-NH2) has been specifically designed for binding of both Zn(II) and Hg(II), i.e. metal ions with different preferences in terms of coordination number, coordination geometry, and to some extent ligand composition. It is demonstrated that HS accommodates both metal ions, and the first coordination sphere, metal ion exchange between peptides, and speciation are characterized as a function of pH using UV-absorption-, synchrotron radiation CD-, (1)H-NMR-, and PAC-spectroscopy as well as potentiometry. Hg(II) binds to the peptide with very high affinity in a {HgS2} coordination geometry, bringing together the two cysteinates close to each end of the peptide in a loop structure. Despite the high affinity, Hg(II) is kinetically labile, exchanging between peptides on the subsecond timescale, as indicated by line broadening in (1)H-NMR. The Zn(II)-HS system displays more complex speciation, involving monomeric species with coordinating cysteinates, histidine, and a solvent water molecule, as well as HS-Zn(II)-HS complexes. In summary, the HS peptide displays conformational flexibility, contains many typical metal ion binding groups, and is able to accommodate metal ions with different structural and ligand preferences with high affinity. As such, the HS peptide may be a scaffold offering binding of a variety of metal ions, and potentially serve for metal ion sequestration in biotechnological applications. PMID:26040991

  17. Competitive adsorption of Cd(II), Zn(II) and Ni(II) from their binary and ternary acidic systems using tourmaline.

    PubMed

    Liu, Haibin; Wang, Cuiping; Liu, Jingting; Wang, Baolin; Sun, Hongwen

    2013-10-15

    The adsorption of Cd(II), Zn(II) and Ni(II) from aqueous solutions in binary and ternary component systems by tourmaline was investigated. Kinetic data were accurately fitted to pseudo-second order and internal diffusion models, which indicated that the adsorption of heavy metals occurred on the interior surface of the sorbent and internal diffusion was the controlling mechanism during heavy metal ion adsorption but was not the only rate-controlling step. Additionally, tourmaline had a very good adsorption capacity for Cd(II), Zn(II) and Ni(II) in multi-component aqueous solutions at strongly acidic pH values (in contrast to industrial wastewater pH values). This good adsorption capacity is attributed to the fact that tourmaline can automatically adjust the pH values of acidic (except pH 2.0 and 3.0), neutral or alkaline aqueous solutions to 6.0. Adsorption isotherms and separation factors showed that tourmaline displays a high selectivity toward one metal in a two-component or a three-component system with an affinity order of Cd(II) > Zn(II) > Ni(II). Thermodynamic parameters indicated that heavy metal adsorption was feasible, spontaneous, and endothermic. Therefore, tourmaline should be explored as a material for removing pollutants from the strongly acidic wastewater. PMID:23851318

  18. Synthesis, spectral and third-order nonlinear optical properties of terpyridine Zn(II) complexes based on carbazole derivative with polyether group

    NASA Astrophysics Data System (ADS)

    Kong, Ming; Liu, Yanqiu; Wang, Hui; Luo, Junshan; Li, Dandan; Zhang, Shengyi; Li, Shengli; Wu, Jieying; Tian, Yupeng

    2015-01-01

    Four novel Zn(II) terpyridine complexes (ZnLCl2, ZnLBr2, ZnLI2, ZnL(SCN)2) based on carbazole derivative group were designed, synthesized and fully characterized. Their photophysical properties including absorption and one-photon excited fluorescence, two-photon absorption (TPA) and optical power limiting (OPL) were further investigated systematically and interpreted on the basis of theoretical calculations (TD-DFT). The influences of different solvents on the absorption and One-Photon Excited Fluorescence (OPEF) spectral behavior, quantum yields and the lifetime of the chromophores have been investigated in detail. The third-order nonlinear optical (NLO) properties were investigated by open/closed aperture Z-scan measurements using femtosecond pulse laser in the range from 680 to 1080 nm. These results revealed that ZnLCl2 and ZnLBr2 exhibited strong two-photon absorption and ZnLCl2 showed superior optical power limiting property.

  19. Crystal structures and thermodynamics/kinetics of Zn(II) coordination polymers with helical chains

    NASA Astrophysics Data System (ADS)

    He, Tian; Yue, Ke-Fen; Zhao, Yi-xing; Chen, San-Ping; Zhou, Chun-sheng; Yan, Ni

    2016-07-01

    Solvothermal reactions of Zn(II) acetates and four V-shaped carboxylates ligands in the presence of 1,4-Bis(2-methyl-imidazol-1-yl)butane afforded four interesting Zn(II) coordination polymers with helical chains, namely, {[Zn(bib)(atibdc)]·2H2O}n (1), {[Zn(bib)(atbip)]·H2O}n (2), [Zn(bib)(2,2‧-tda)]}n (3) and {[Zn(bib)(5-tbipa)]·EtOH}n (4), (H2atibdc=5-amino-2,4,6-triiodoisophthalic acid, H2atbip=5-amino-2,4,6-tribromoisophthalic acid, 2,2‧-H2tad=2,2‧-thiodiacetic acid, 5-H2tbipa=5-tert-butyl-isophthalic acid). 1 reveals a 3D chiral framework with three kinds of helical chains along a, b and c axis. 2 shows a 2D step-type chiral framework with right-handed helical chains. 3 displays a wavelike 2D layer network possessing alternate left- and right-handed helical chains. 4 presents a four-connected 3D framework with zigzag and meso-helical chains. The different spacers and substituent group of carboxylic acid ligands may lead to the diverse network structures of 1-4. The fluorescent properties of complexes 1-4 were studied. In addition, the thermal decompositions properties of 1-4 were investigated by simultaneous TG/DTG-DSC technique. The apparent activation energy E and the pre-exponential factor (A) of skeleton collapse for the complexes 1-4 are calculated by the integral Kissinger's method and Ozawa-Doyle's method. The activation energy E (E1=209.658 kJ·mol-1, E2=250.037 kJ mol-1, E3=225.300 kJ mol-1, E4=186.529 kJ·mol-1) demonstrates that the reaction rate of the melting decomposition is slow. The thermodynamic parameters (ΔH‡, ΔG‡ and ΔS‡) at the peak temperatures of the DTG curves were also calculated. ΔG‡>0 indicates that the skeleton collapse is not spontaneous. ΔHd>0 suggests that the skeleton collapse is endothermic, corresponding to the intense endothermic peak of the DSC curve. The structural stability could be illustrated from the point of thermodynamics and kinetics. Their thermal decompositions properties of 1-4 were

  20. Adsorption and Precipitation of Aqueous Zn(II) on Hematite Nano- and Microparticles

    SciTech Connect

    Ha, Juyong; Farges, Francois; Brown, Gordon E., Jr.; /SLAC, SSRL

    2006-12-13

    As part of a study of the effect of particle size on reactivity of hematite to aqueous metal ions, the sorption of Zn(II) on hematite nanoparticles and microparticles was examined over a wide range of Zn(II) concentrations using Zn K-edge EXAFS. When reacted with nanoparticles at pH 5.5 and low Zn(II) sorption densities (0.04 {le} {Lambda} < 2.76 imol/m{sup 2}), Zn(II) formed five-coordinated or a mixture of four- and six-coordinated surface complexes with an average Zn-O distance of 2.04({+-}0.02){angstrom}. At pH 5.5 and high Zn(II) sorption densities (2.76 {ge} {Lambda} {le} 3.70 mol/m{sup 2}), formation of surface precipitates is suggested based on the presence of second-shell Zn and multiple scattering features in the Fourier transform (FT) of the EXAFS spectra. EXAFS fitting of these high {Lambda} samples yielded an average first-shell Zn-O distance of 2.10({+-}0.02){angstrom}, with second-shell Zn-Fe and Zn-Zn distances of 3.23({+-}0.03){angstrom} and 3.31({+-}0.03){angstrom}, respectively. Qualitative comparison between the EXAFS spectra of these sorption samples and that of amorphous zinc hydroxide and Zn-bearing hydrotalcite indicates the development of surface precipitates with increasing {Lambda}. EXAFS spectra of Zn(II) sorbed on hematite microparticles under similar experimental conditions showed no evidence for surface precipitates even at the highest Zn surface coverage ({Lambda} = 4 {micro}mol/m{sup 2}). These results indicate that reactivities of hematite nanoparticles and macroparticles differ with respect to Zn(II)aq, depending on Zn(II) sorption density. We suggest that the degree of hematite crystallinity affects the reactivity of hematite surfaces toward Zn(II)aq and the formation of the Zn(II) surface complexes.

  1. Characterization of the Zn(II) Binding Properties of the Human Wilms’ Tumor Suppressor Protein C-terminal Zinc Finger Peptide

    PubMed Central

    2015-01-01

    Zinc finger proteins that bind Zn(II) using a Cys2His2 coordination motif within a ββα protein fold are the most abundant DNA binding transcription factor domains in eukaryotic systems. These classic zinc fingers are typically unfolded in the apo state and spontaneously fold into their functional ββα folds upon incorporation of Zn(II). These metal-induced protein folding events obscure the free energy cost of protein folding by coupling the protein folding and metal-ion binding thermodynamics. Herein, we determine the formation constant of a Cys2His2/ββα zinc finger domain, the C-terminal finger of the Wilms’ tumor suppressor protein (WT1-4), for the purposes of determining its free energy cost of protein folding. Measurements of individual conditional dissociation constants, Kd values, at pH values from 5 to 9 were determined using fluorescence spectroscopy by direct or competition titration. Potentiometric titrations of apo-WT1-4 followed by NMR spectroscopy provided the intrinsic pKa values of the Cys2His2 residues, and corresponding potentiometric titrations of Zn(II)–WT1-4 followed by fluorescence spectroscopy yielded the effective pKaeff values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKaeff values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)–WT1-4, KfML value of 7.5 × 1012 M–1, with a limiting Kd value of 133 fM. This shows that Zn(II) binding to the Cys2His2 site in WT1-4 provides at least −17.6 kcal/mol in driving force to fold the protein scaffold. A comparison of the conditional dissociation constants of Zn(II)–WT1-4 to those from the model peptide Zn(II)–GGG–Cys2His2 over the pH range 5.0 to 9.0 and a comparison of their pH-independent KfML values demonstrates that the free energy cost of protein folding in WT1-4 is less than +2.1 kcal/mol. These results validate our GGG model system for determining the cost of protein folding in natural

  2. Special Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Parikh, Indu; Cuatrecasas, Pedro

    1985-01-01

    Describes the nature of affinity chromatography and its use in purifying enzymes, studying cell interactions, exploring hormone receptors, and other areas. The potential the technique may have in treating disease is also considered. (JN)

  3. Adsorption of Co(II), Ni(II), Cu(II), and Zn(II) on hexagonal templated zirconia obtained thorough a sol-gel process: the effects of nanostructure on adsorption features.

    PubMed

    de Farias, Robson F; do Nascimento, Ana A S; Bezerra, Cícero W B

    2004-09-01

    Using zirconium tetrabutoxide, diaminedecane, and diamineoctane as precursors, a templated hexagonal zirconia matrix is synthesized and characterized by X-ray diffractometry and scanning electron microscopy. The adsorption capacity of such a matrix toward Co(II), Ni(II), Cu(II), and Zn(II) from aqueous solutions is studied. The adsorption affinity of the synthesized hexagonal templated zirconia toward the cations is Cu(II)>Zn(II) >Ni(II)>Co(II). It is also verified that the adsorption of the cations follows a Langmuir and not a Freundlich isotherm. All obtained isotherms are of type I, according to the IUPAC classification. The observed adsorption affinity sequence can be explained by taking into account the velocity constant for the substitution of water molecules into the cation coordination spheres, as well as the Irving-Williams series. PMID:15276032

  4. Cu(II) and Zn(II) adsorption capacity of three different clay liner materials.

    PubMed

    Musso, T B; Parolo, M E; Pettinari, G; Francisca, F M

    2014-12-15

    Sorption of Cu(II) and Zn(II) on three natural clays meeting the international requirements for use as liners was evaluated by means of batch tests. The purpose of this research was to determine the retention capacities of the clays for metal cations commonly present in urban solid waste leachates. The pH and ionic strength conditions were set at values frequently found in real leachates. The changes observed in the XRD patterns and FTIR spectra upon adsorption can be considered an evidence of clay-metal electrostatic interaction. The Langmuir model was found to best describe the sorption processes, offering maximum sorption capacities from 8.16 to 56.89 mg/g for Cu(II) and from 49.59 to 103.83 mg/g for Zn(II). All samples remove more Zn(II) than Cu(II), which may be related to the different geometry of the hydrated Cu(II) cation. The total amount of metal sorption was strongly influenced by the total specific surface area, the presence of carbonates and the smectite content of the clays. In addition to their known quality as physical barriers, the adsorbed amounts obtained indicate the suitability of the tested clays to contribute to the retardation of Cu(II) and Zn(II) transport through clay liners. PMID:25156265

  5. BIOCHEMISTRY OF MOBILE ZINC AND NITRIC OXIDE REVEALED BY FLUORESCENT SENSORS

    PubMed Central

    Pluth, Michael D.; Tomat, Elisa; Lippard, Stephen J.

    2010-01-01

    Biologically mobile zinc and nitric oxide (NO) are two prominent examples of inorganic compounds involved in numerous signaling pathways in living systems. In the past decade, a synergy of regulation, signaling, and translocation of these two species has emerged in several areas of human physiology, providing additional incentive for developing adequate detection systems for Zn(II) ions and NO in biological specimens. Fluorescent probes for both of these bioinorganic analytes provide excellent tools for their detection, with high spatial and temporal resolution. We review the most widely used fluorescent sensors for biological zinc and nitric oxide, together with promising new developments and unmet needs of contemporary Zn(II) and NO biological imaging. The interplay between zinc and nitric oxide in the nervous, cardiovascular, and immune systems is highlighted to illustrate the contributions of selective fluorescent probes to the study of these two important bioinorganic analytes. PMID:21675918

  6. Interaction of Aqueous Zn(II) with Hematite Nanoparticles and Microparticles. Part 1. EXAFS Study of Zn(II) Adsorption and Precipitation

    SciTech Connect

    Ha, Juyoung; Trainor, Thomas P.; Farges, Francois; Brown, Jr., Gordon E.

    2009-06-30

    Sorption of Zn(II){sub (aq)} on hematite (a-Fe{sub 2}O{sub 3}) nanoparticles (average diameter 10.5 nm) and microparticles (average diameter 550 nm) has been examined over a range of total Zn(II){sub (aq)} concentrations (0.4-7.6 mM) using Zn K-edge EXAFS spectroscopy and selective chemical extractions. When ZnCl{sub 2} aqueous solutions were reacted with hematite nanoparticles (HN) at pH 5.5, Zn(II) formed a mixture of four- and six-coordinated surface complexes [Zn(O,OH){sub 4} and Zn(O,OH){sub 6}] with an average Zn-O distance of 2.04 {+-} 0.02 {angstrom} at low sorption densities ({Gamma} {<=} 1.1 {mu}mol/m{sup 2}). On the basis of EXAFS-derived Zn-Fe{sup 3+} distances of (3.10-3.12) {+-} 0.02 {angstrom}, we conclude that both Zn(O,OH){sub 6} and Zn(O,OH){sub 4} adsorb on octahedral Fe{sup 3+}(O,OH){sub 6} or pentahedral Fe{sup 3+}(O,OH){sub 5} surface sites on HN as inner-sphere, mononuclear, bidentate, edge-sharing adsorption complexes at these low sorption densities. It is possible that polynuclear Zn complexes are also present because of the similarity of Zn and Fe backscattering. At higher Zn(II) sorption densities on hematite nanoparticles ({Gamma} {>=} 3.38 {mu}mol/m{sup 2}), we observed the formation of Zn(O,OH){sub 6} surface complexes, with an average Zn-O distance of 2.09 {+-} 0.02 {angstrom}, a Zn-Zn distance of 3.16 {+-} 0.02 {angstrom}, and a linear multiple-scattering feature at 6.12 {+-} 0.06 {angstrom}. Formation of a Zn(OH){sub 2(am)} precipitate for the higher sorption density samples ({Gamma} {>=} 3.38 {mu}mol/m{sup 2}) is suggested on the basis of comparison of the EXAFS spectra of the sorption samples with that of synthetic Zn(OH){sub 2(am)}. In contrast, EXAFS spectra of Zn(II) sorbed on hematite microparticles (HM) under similar experimental conditions showed no evidence of surface precipitates even at the same total [Zn(II){sub (aq)}] that resulted in precipitate formation in the nanoparticle system. Instead, Zn(O,OH){sub 6} octahedra

  7. Two new Zn(II) and Cd(II) coordinastion polymers based on amino-tetrazole and phenylcarboxylate: Syntheses, topological structures and photoluminescent properties

    SciTech Connect

    Liu, Dong-Sheng; College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou, Fujian 350108 ; Sui, Yan; Chen, Weng-Tong; Huang, Jian-Gen; Chen, Jian-Zhong; Huang, Chang-Cang

    2012-12-15

    Two Zn(II) and Cd(II) compounds with the in-situ generated ligand of 5-amino-tetrazolate (atz{sup -}) were prepared from the hydrothermal reactions of the corresponding Cd or Zn(II) salts with phenylcarboxylate, and characterized by elemental analysis, IR spectroscopy, and TGA. The results of X-ray crystallographic analysis reveal that compound [Zn{sub 2}(BZA)(atz){sub 2}(OH)]{sub n} (1) (BZA=benzoic acid) presents a two-dimensional (2D) 'hcb' topological network constructed from the ZnN{sub 2}O{sub 2} tetrahedra. In compound [Cd{sub 6}(atz){sub 6}(PTA){sub 3}]{sub n} (2) (PTA=terephthalic acid), the identical [Cd{sub 3}(atz){sub 3})]{sup 3+}{sub n} clusters are connected by atz ligands to generate a 2D cationic layer, and the neighboring cationic layers are pillared by PTA giving birth to 3D network. After simplifying, the complicated 3D network of 2 can be presented as an unprecedented (4, 4, 10)-connected trinodal topology. The formations of the structures show a good example that using the combination of the in-situ generated ligand and other coligand synthetic strategy can construct interesting topological structures. The thermal stabilities and fluorescent properties of the complexes have also been studied. - Graphical abstract: Two d{sup 10} metal complexes have been synthesized by employing mixed-ligand synthetic approach. Complex 1 presents a 2D 'hcb' topological network. Complex 2 shows an unprecedented (4, 4, 10)-connected trinodal topology. Highlights: Black-Right-Pointing-Pointer Coligand synthetic strategy was applied to obtain new MOFs with useful properties. Black-Right-Pointing-Pointer Two new Zn(II) and Cd(II) complexes were constructed from the mixed-ligand. Black-Right-Pointing-Pointer Topologically, compound 2 presented an unprecedented (4, 4, 10)-connected trinodal topology. Black-Right-Pointing-Pointer The two compounds may be excellent candidates for potential photoactive material.

  8. Preliminary study of the metal binding site of an anti-DTPA-indium antibody by equilibrium binding immunoassays and immobilized metal ion affinity chromatography.

    PubMed

    Boden, V; Colin, C; Barbet, J; Le Doussal, J M; Vijayalakshmi, M

    1995-01-01

    Creating metal coordination sites by modifying an existing enzyme or by eliciting antibodies against metal chelate haptens is of great interest in biotechnology to create enzyme catalysts with novel specificities. Here, we investigate the metal binding potential of a monoclonal antibody raised against a DTPA-In(III) hapten (mAb 734). We study its relative binding efficiency to metals of biological relevance by equilibrium binding immunoassays and immobilized metal ion affinity chromatography, two approaches which can give complementary information regarding composition and/or structure of the metal binding site(s). Fe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(II) binding was compared to In(III). All of them were shown to displace indium, but their affinity for mAb 734 decreased by 100-fold compared to indium. Competitive metal binding immunoassays between Zn(II) and In(III) revealed an unusual behavior by Zn(II) which remains to be explained. Moreover, IMAC allowed us to predict the metal binding amino acids involved in the antibody paratope. The antibody metal binding site was shown to contain at least two histidine residues in a cluster, and the presence of aspartic and glutamic acid as well as cysteine residues could not be excluded. Thus, simple competition studies allows us to obtain some partial information on the metal binding structural features of this anti-metal chelate antibody and to guide our screening of its catalytic potential. PMID:7578356

  9. Intracellular distribution of fluorescent copper and zinc bis(thiosemicarbazonato) complexes measured with fluorescence lifetime spectroscopy.

    PubMed

    Hickey, James L; James, Janine L; Henderson, Clare A; Price, Katherine A; Mot, Alexandra I; Buncic, Gojko; Crouch, Peter J; White, Jonathan M; White, Anthony R; Smith, Trevor A; Donnelly, Paul S

    2015-10-01

    The intracellular distribution of fluorescently labeled copper and zinc bis(thiosemicarbazonato) complexes was investigated in M17 neuroblastoma cells and primary cortical neurons with a view to providing insights into the neuroprotective activity of a copper bis(thiosemicarbazonato) complex known as Cu(II)(atsm). Time-resolved fluorescence measurements allowed the identification of the Cu(II) and Zn(II) complexes as well as the free ligand inside the cells by virtue of the distinct fluorescence lifetime of each species. Confocal fluorescent microscopy of cells treated with the fluorescent copper(II)bis(thiosemicarbazonato) complex revealed significant fluorescence associated with cytoplasmic puncta that were identified to be lysosomes in primary cortical neurons and both lipid droplets and lysosomes in M17 neuroblastoma cells. Fluorescence lifetime imaging microscopy confirmed that the fluorescence signal emanating from the lipid droplets could be attributed to the copper(II) complex but also that some degree of loss of the metal ion led to diffuse cytosolic fluorescence that could be attributed to the metal-free ligand. The accumulation of the copper(II) complex in lipid droplets could be relevant to the neuroprotective activity of Cu(II)(atsm) in models of amyotrophic lateral sclerosis and Parkinson's disease. PMID:26397162

  10. Adsorption of enrofloxacin in presence of Zn(II) on a calcareous soil.

    PubMed

    Graouer-Bacart, Mareen; Sayen, Stéphanie; Guillon, Emmanuel

    2015-12-01

    As a result of their consumption, excretion, disposal and persistence, antibiotics enter the soil environment and may be transported to surface and ground waters. During their transfer through soils, retention processes play a key role in their mobility. Antibiotics often coexist with heavy metals in soils due to agricultural practices and other sources of inputs. In this context, this study deals with the co-adsorption of Zn(II) and enrofloxacin (ENR), a widely-used veterinary antibiotic, on a calcareous soil using batch retention experiments and X-ray Absorption Near Edge Structure (XANES) spectroscopy. To improve our understanding of the interaction of this emerging organic contaminant with metal cations at the water-soil interface, the ternary system containing ENR, Zn(II) and a selected calcareous soil was investigated over a pH range between 7 and 10, at different solid-solution contact times and ENR concentrations. The presence of Zn(II) slightly influenced the retention of the antibiotic, leading to an increase of the adsorbed ENR amounts. The distribution coefficient Kd value increased from 0.66 Lg(-1) for single ENR adsorption to 1.04 Lg(-1) in presence of Zn(II) at a 1/2 ENR/Zn(II) ratio. The combination of adsorption isotherm data, solution speciation diagrams and XANES spectra evidenced a small proportion of Zn(II)-ENR complexes at soil pH leading to the slight increase of ENR adsorption in presence of zinc. These results suggest that it is necessary to consider the interaction between ENR and metal cations when assessing the mobility of ENR in soils. PMID:26408826

  11. Mapping Protein–Protein Interactions of the Resistance-Related Bacterial Zeta Toxin–Epsilon Antitoxin Complex (ε2ζ2) with High Affinity Peptide Ligands Using Fluorescence Polarization

    PubMed Central

    Fernández-Bachiller, María Isabel; Brzozowska, Iwona; Odolczyk, Norbert; Zielenkiewicz, Urszula; Zielenkiewicz, Piotr; Rademann, Jörg

    2016-01-01

    Toxin–antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta–Epsilon toxin–antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε2ζ2 complex. Three α helices of Zeta forming the protein–protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε2ζ2 complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay. PMID:27438853

  12. Mapping Protein-Protein Interactions of the Resistance-Related Bacterial Zeta Toxin-Epsilon Antitoxin Complex (ε₂ζ₂) with High Affinity Peptide Ligands Using Fluorescence Polarization.

    PubMed

    Fernández-Bachiller, María Isabel; Brzozowska, Iwona; Odolczyk, Norbert; Zielenkiewicz, Urszula; Zielenkiewicz, Piotr; Rademann, Jörg

    2016-01-01

    Toxin-antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta-Epsilon toxin-antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε₂ζ₂ complex. Three α helices of Zeta forming the protein-protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε₂ζ₂ complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay. PMID:27438853

  13. RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking.

    PubMed

    Dolgosheina, Elena V; Jeng, Sunny C Y; Panchapakesan, Shanker Shyam S; Cojocaru, Razvan; Chen, Patrick S K; Wilson, Peter D; Hawkins, Nancy; Wiggins, Paul A; Unrau, Peter J

    2014-10-17

    Because RNA lacks strong intrinsic fluorescence, it has proven challenging to track RNA molecules in real time. To address this problem and to allow the purification of fluorescently tagged RNA complexes, we have selected a high affinity RNA aptamer called RNA Mango. This aptamer binds a series of thiazole orange (fluorophore) derivatives with nanomolar affinity, while increasing fluorophore fluorescence by up to 1,100-fold. Visualization of RNA Mango by single-molecule fluorescence microscopy, together with injection and imaging of RNA Mango/fluorophore complex in C. elegans gonads demonstrates the potential for live-cell RNA imaging with this system. By inserting RNA Mango into a stem loop of the bacterial 6S RNA and biotinylating the fluorophore, we demonstrate that the aptamer can be used to simultaneously fluorescently label and purify biologically important RNAs. The high affinity and fluorescent properties of RNA Mango are therefore expected to simplify the study of RNA complexes. PMID:25101481

  14. Visualizing the kinetic power stroke that drives proton-coupled Zn(II) transport

    PubMed Central

    Gupta, Sayan; Chai, Jin; Cheng, Jie; D'Mello, Rhijuta; Chance, Mark R.; Fu, Dax

    2014-01-01

    The proton gradient is a principal energy source for respiration-dependent active transport, but the structural mechanisms of proton-coupled transport processes are poorly understood. YiiP is a proton-coupled zinc transporter found in the cytoplasmic membrane of E. coli, and the transport-site of YiiP receives protons from water molecules that gain access to its hydrophobic environment and transduces the energy of an inward proton gradient to drive Zn(II) efflux1,2. This membrane protein is a well characterized member3-7 of the protein family of cation diffusion facilitators (CDFs) that occurs at all phylogenetic levels8-10. X-ray mediated hydroxyl radical labeling of YiiP and mass spectrometric analysis showed that Zn(II) binding triggered a highly localized, all-or-none change of water accessibility to the transport-site and an adjacent hydrophobic gate. Millisecond time-resolved dynamics revealed a concerted and reciprocal pattern of accessibility changes along a transmembrane helix, suggesting a rigid-body helical reorientation linked to Zn(II) binding that triggers the closing of the hydrophobic gate. The gated water access to the transport-site enables a stationary proton gradient to facilitate the conversion of zinc binding energy to the kinetic power stroke of a vectorial zinc transport. The kinetic details provide energetic insights into a proton-coupled active transport reaction. PMID:25043033

  15. New hybrid materials as Zn(II) sorbents in water samples

    SciTech Connect

    Perez-Quintanilla, Damian

    2010-09-15

    Mesoporous silicas have been chemically modified with 5-mercapto-1-methyltetrazole (MTTZ) obtaining hybrid materials denominated MTTZ-MSU-2 and MTTZ-HMS. These materials were employed as Zn(II) sorbents from aqueous media at room temperature. The effect of several variables (stirring time, pH, presence of other metals) has been studied using batch and column techniques. Flame atomic absorption spectrometry (FAAS) was used to determinate Zn(II) concentration in the filtrate or in the eluted solution after the adsorption process. The results indicate that under pH 8, the maximum adsorption value was 0.94 {+-} 0.01 and 0.72 {+-} 0.01 mmol Zn(II)/g for MTTZ-MSU-2 and MTTZ-HMS, respectively. In tap water samples, a preconcentration factor of 200 was obtained. On the basis of these results, it can be concluded that it is possible to modify chemically MSU-2 and HMS with 5-mercapto-1-methyltetrazole and to use the resulting modified mesoporous silica as an effective adsorbent for Zn(II) in aqueous media.

  16. A novel Zn(II) complex of N-nicotinyl phosphoramide: Combined experimental and computational studies

    NASA Astrophysics Data System (ADS)

    Gholivand, Khodayar; Molaei, Foroogh; Thibonnet, Jérôme

    2015-07-01

    A novel Zn(II) coordination polymer of N-nicotinyl phosphoric triamide ligand, {[Zn(L)2(H2O)2]ṡ(NO3)2}n C1 (L = 3-NC5H4C(O)NHP(O)(NC6H12)2), has been synthesized and characterized by IR and 1H, 13C, 31P NMR spectroscopy. Crystal structure analysis of C1 demonstrates a distorted octahedral geometry for Zn(II) ions. The oxygen atom of phosphoryl group (Ophosphoryl) and the nitrogen atom of pyridine ring (Npyridine) of ligand take part in coordination to Zn(II) centers in a bidentate bridging mode. This coordination pattern results in infinite 1D polymeric chains along c axis, which are composed of metal shared 16-membered puckered rings. Structural data show that despite binding through Ophosphoryl, the Pdbnd O bond distance unexpectedly shortens in C1 when compared to that of the ligand, conceivably due to the steric factors in the solid state. This is confirmed by comparing the structural and electronic properties of C1 and L in the gas phase by using density functional theory (DFT) calculations. Natural bond orbital (NBO) analysis reveals the metal-ligand interaction for C1 as donor-acceptor type delocalizations (charge transfer). Besides, on the basis of the quantum theory of atoms in molecules (QTAIM) analysis, the nature of Znsbnd O and Znsbnd N bondings is found to be mainly electrostatic with a small amount of covalent character.

  17. Local Affinity Release.

    PubMed

    Delplace, Vianney; Obermeyer, Jaclyn; Shoichet, Molly S

    2016-07-26

    The use of hydrogels for therapeutic delivery is a burgeoning area of investigation. These water-swollen polymer matrices are ideal platforms for localized drug delivery that can be further combined with specific ligands or nanotechnologies to advance the controlled release of small-molecule drugs and proteins. Due to the advantage of hydrophobic, electrostatic, or specific extracellular matrix interactions, affinity-based strategies can overcome burst release and challenges associated with encapsulation. Future studies will provide innovative binding tools, truly stimuli-responsive systems, and original combinations of emerging technologies to control the release of therapeutics spatially and temporally. Local drug delivery can be achieved by directly injecting a therapeutic to its site of action and is advantageous because off-target effects associated with systemic delivery can be minimized. For prolonged benefit, a vehicle that provides sustained drug release is required. Hydrogels are versatile platforms for localized drug release, owing to the large library of biocompatible building blocks from which they can be formed. Injectable hydrogel formulations that gel quickly in situ and provide sustained release of therapeutics are particularly advantageous to minimize invasiveness. The incorporation of polymers, ligands or nanoparticles that have an affinity for the therapeutic of interest improve control over the release of small-molecule drugs and proteins from hydrogels, enabling spatial and temporal control over the delivery. Such affinity-based strategies can overcome drug burst release and challenges associated with protein instability, allowing more effective therapeutic molecule delivery for a range of applications from therapeutic contact lenses to ischemic tissue regeneration. PMID:27403513

  18. Morphometric affinities of gigantopithecus.

    PubMed

    Gelvin, B R

    1980-11-01

    Multivariate analyses, supplemented by univariate statistical methods, of measurements from mandibular tooth crown dimensions and the mandible of Gigantopithecus blacki, G. bilaspurensis, Plio-Plelstocene hominids, Homo erectus, and seven Neogene ape species from the genera Proconsul, Sivapithecus, Ouranopithecus, and Dryopithecus were used to assess the morphometric affinities of Gigantopithecus. The results show that Gigantopithecus displays affinities to Ouranopithecus and to the hominids, particularly the Plio-Plelstocene hominids, rather than to the apes. Ouranopithecus demonstrated dental resemblances to G. bilaspurensis and the Plio-Pleistocene hominids but mandibular similarities to the apes. Results of analyses of tooth and mandibular shape indices, combined with multivariate distance and temporal relationships, suggest that Ouranopithecus is a more likely candidate for Gigantopithecus ancestry than is Silvapithecus indicus. Shape and allometric differences between G. bilaspurensis and the robust australopithecines weaken the argument for an ancestral-descendant relationship between these groups. The results support the hypothesis that Gigantopithecus is an extinct side branch of the Hominidae. PMID:7468790

  19. Synthetic hydroxyapatites doped with Zn(II) studied by X-ray diffraction, infrared, Raman and thermal analysis

    NASA Astrophysics Data System (ADS)

    Guerra-López, José R.; Echeverría, Gustavo A.; Güida, Jorge A.; Viña, Raúl; Punte, Graciela

    2015-06-01

    Calcium hydroxyapatite (CaHap) formation when different amounts of Zn(II) are present in the mother solution has been investigated by atomic absorption, infrared and Raman spectroscopies, X-ray diffraction and thermal analysis (DTA and TG). The studied samples have been synthesized at T=95 °C and pH 9 in air. The analysis of the results have shown that the pure CaHap sample crystallizes in the monoclinic form P21/b. Concentrations up to 20% of Zn(II) in the mother solution, equivalent to smaller concentrations in solid (up to 9.1% in wt), favor the formation of the hexagonal apatite, P63/m, while Zn(II) concentrations higher than 20% in solution help an amorphous phase development where vibrational spectra indicated coexistence of two phases: an apatite and ZnNH4PO4·H2O. Infrared data of thermal treated samples endorse that HPO42- ion had not been incorporated in Zn(II) doped samples during the synthesis process. Present results also allow to conclude that Zn(II) cation exhibits a preference to occupy the Ca2 site of the apatite structure and induces water adsorption and a small quantity of CO32- cation incorporation, leading to formation of a less crystalline Ca deficient apatite.

  20. Removal of Pb(II) and Zn(II) from Aqueous Solutions by Raw Crab Shell: A Comparative Study.

    PubMed

    Zhou, Chuanqiang; Gong, Xiangxiang; Han, Jie; Guo, Rong

    2016-04-01

    Removals of Pb(II) and Zn(II) ions from water using crab (Clistocoeloma sinensis) shell particles as biosorbent have been compared in this study. Uptake equilibriums for two ions well described by Langmuir isotherm revealed that crab shell possessed higher uptake capacity for Pb(II) (709 mg/g) than that for Zn(II) (117 mg/g). Kinetics data for the uptake of the two metals were successfully modeled using the pseudo-second-order model, where the initial uptake rate of Pb(II) was much faster than that of Zn(II). Dubinin-Radushkevick modeling and thermodynamic parameters hinted at different uptake mechanisms of Pb(II) and Zn(II) removal by crab shell, attested by FTIR, XRD, FESEM analysis. Pb(II) ion was removed mainly through the chemical reaction, while the uptake of Zn(II) ion onto crab shell was attributed to the chelation and coordination interactions. The polluted river water and laboratory wastewater both satisfied the standards for drinking and irrigation/fishery water, respectively, after being treated with crab shell particles. PMID:26864343

  1. Adjoint affine fusion and tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  2. Multifunctional Zn(II) Complexes: Photophysical Properties and Catalytic Transesterification toward Biodiesel Synthesis.

    PubMed

    Gupta, Abhishek Kumar; Dhir, Abhimanew; Pradeep, Chullikkattil P

    2016-08-01

    Using 4-substituted derivatives of phenol-based compartmental Schiff-base hydroxyl-rich ligand, four multifunctional binuclear Zn(II) complexes have been synthesized and characterized. The photophysical properties of these complexes were explored in the solid state, in solutions, and in poly(methyl methacrylate) (PMMA) matrix, which revealed their good potential as tunable solid state emitters. Some of these complexes acted as efficient catalysts for the transesterification of esters and canola oil showing their potential in biodiesel generation. Mechanistic investigations using ESI-MS revealed that the transesterification catalyzed by these complexes proceeds through two types of acyl intermediates. PMID:27439021

  3. Coordination structure of adsorbed Zn(II) at Water-TiO2 interfaces

    SciTech Connect

    He, G.; Pan, G.; Zhang, M.; Waychunas, G.A.

    2011-01-15

    The local structure of aqueous metal ions on solid surfaces is central to understanding many chemical and biological processes in soil and aquatic environments. Here, the local coordination structure of hydrated Zn(II) at water-TiO{sub 2} interfaces was identified by extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) spectroscopy combined with density functional theory (DFT) calculations. A nonintegral coordination number of average {approx}4.5 O atoms around a central Zn atom was obtained by EXAFS analysis. DFT calculations indicated that this coordination structure was consistent with the mixture of 4-coordinated bidentate binuclear (BB) and 5-coordinated bidentate mononuclear (BM) metastable equilibrium adsorption (MEA) states. The BB complex has 4-coordinated Zn, while the monodentate mononuclear (MM) complex has 6-coordinated Zn, and a 5-coordinated adsorbed Zn was found in the BM adsorption mode. DFT calculated energies showed that the lower-coordinated BB and BM modes were thermodynamically more favorable than the higher-coordinated MM MEA state. The experimentally observed XANES fingerprinting provided additional direct spectral evidence of 4- and 5-coordinated Zn-O modes. The overall spectral and computational evidence indicated that Zn(II) can occur in 4-, 5-, and 6-oxygen coordinated sites in different MEA states due to steric hindrance effects, and the coexistence of different MEA states formed the multiple coordination environments.

  4. Affinity chromatography: a historical perspective.

    PubMed

    Hage, David S; Matsuda, Ryan

    2015-01-01

    Affinity chromatography is one of the most selective and versatile forms of liquid chromatography for the separation or analysis of chemicals in complex mixtures. This method makes use of a biologically related agent as the stationary phase, which provides an affinity column with the ability to bind selectively and reversibly to a given target in a sample. This review examines the early work in this method and various developments that have lead to the current status of this technique. The general principles of affinity chromatography are briefly described as part of this discussion. Past and recent efforts in the generation of new binding agents, supports, and immobilization methods for this method are considered. Various applications of affinity chromatography are also summarized, as well as the influence this field has played in the creation of other affinity-based separation or analysis methods. PMID:25749941

  5. Removal of Zn(II) from electroplating effluent using yeast biofilm formed on gravels: batch and column studies

    PubMed Central

    2014-01-01

    Background Present study deals with the removal of Zn(II) ions from effluent using yeast biofilm formed on gravels. Methods The biofilm forming ability of Candida rugosa and Cryptococcus laurentii was evaluated using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay and monitored by scanning electron microscopy (SEM), and Confocal laser scanning microscopy (CLSM). Copious amount of extracellular polymeric substances (EPS) produced by yeast species was quantified and characterized by Fourier transform infrared spectroscopy (FT-IR). Results Yeast biofilm formed on gravels by C. rugosa and C. laurentii showed 88% and 74.2% removal of Zn(II) ions respectively in batch mode. In column mode, removal of Zn(II) ions from real effluent was found to be 95.29% by C. rugosa biofilm formed on gravels. Conclusion The results of the present study showed that there is a scope to develop a cost effective method for the efficient removal of Zn(II) from effluent using gravels coated with yeast biofilm. PMID:24397917

  6. Fluorescent Aptamer Sensors

    NASA Astrophysics Data System (ADS)

    Chen, Hui William; Kim, Youngmi; Meng, Ling; Mallikaratchy, Prabodhika; Martin, Jennifer; Tang, Zhiwen; Shangguan, Dihua; O'Donoghue, Meghan; Tan, Weihong

    Aptamers are single-stranded nucleic acid probes that can be evolved to have high specificity and affinity for different targets. These targets include biomar-ker proteins, small molecules, and even whole live cells that express a variety of surface proteins of interest. Aptamers offer several advantages over protein-based molecular probes such as low immunogenic activity, flexible modification, and in vitro synthesis. In addition, aptamers used as molecular probes can be made with easy signaling for binding with their corresponding targets. There are a few different fluorescence-based signal transduction mechanisms, such as direct fluorophore labeling, fluorescence resonance energy transfer (FRET), fluorescence quenching, fluorescence anisotropy, and light-switching excimers. These signaling processes in combination with various labeling strategies of nucleic acid aptamers contribute to simple, rapid, sensitive, and selective biological assays. In this chapter, we discuss the optical signaling of aptamers for single proteins such as α-thrombin and platelet-derived growth factor (PDGF). We also present detailed discussion about fluorescent aptamers developed from cell-based systematic evolution of ligands by exponential enrichment (SELEX) for the recognition of different target tumor cells.

  7. The pH dependent Cu(II) and Zn(II) binding behavior of an analog methanobactin peptide.

    PubMed

    Sesham, Ramakrishna; Choi, Dongwon; Balaji, Anupama; Cheruku, Sahithi; Ravichetti, Chiranjeevi; Alsharani, Aisha A; Nasani, Mahesbabu; Angel, Laurence A

    2013-01-01

    The pH dependent reactivity of an analog methanobactin peptide (amb) with the sequence acetyl-His1-Cys2-Gly3-Pro4-His5-Cys6 (Mw = 694.79 Da) was investigated for its binding ability for a series of biologically active metal ions using ion mobility-mass spectrometry. Cu(II), Zn(II) and, to a lesser extent, Ni(II) were observed to form complexes with amb from 1 : 1 molar equivalent amb:metal(II) solutions at pH > 6, indicating the deprotonation of the imidazole N of His (pKa = 6.0) must occur to allow the initial anchoring of the metal(II) ion. The amb-metal(II) complexes were observed as both positive and negative ions, although the Zn(II) complexes preferred forming an overall negative ion complex which is consistent with the two thiolate groups of Cys2 and Cys6 being involved in Zn(II) coordination. The Cu(II) addition, however, always resulted in a Cys-Cys disulfide bridge in both Cu-free amb and Cu-bound amb, which excluded thiolate coordination to Cu(II). Collision cross- section measurements showed the Zn(II) and Cu(II) negative ion complexes were smaller than the positive ion complexes, suggesting Zn(II) binds most compactly via the imidazole N of His and the thiolate groups of Cys, whereas Cu(II) binds most compactly via the imidazole N of His and two deprotonated N of two amide groups on the peptide backbone. The lowest energy structures from the B3LYP/LanL2DZ level of theory showed the functional groups of His5, Cys2 and Cys6 coordinated to Zn(II), whereas the His1 and the amide nitrogens of Cys2 and Gly3 coordinated to Cu(II), producing an overall negative charged complex. The positive ion complexes of Zn(II) and Cu(II) were both shown to coordinate via the two imidazole nitrogens of His1 and His5 and either the oxygen of the backbone carbonyl of Cys6 or the oxygen of the C-terminal, respectively. PMID:24378464

  8. Recent advances in affinity capillary electrophoresis for binding studies.

    PubMed

    Albishri, Hassan M; El Deeb, Sami; AlGarabli, Noura; AlAstal, Raghda; Alhazmi, Hassan A; Nachbar, Markus; El-Hady, Deia Abd; Wätzig, Hermann

    2014-01-01

    The present review covers recent advances and important applications of affinity capillary electrophoresis (ACE). It provides an overview about various ACE types, including ACE-MS, the multiple injection mode, the use of microchips and field-amplified sample injection-ACE. The most common scenarios of the studied affinity interactions are protein-drug, protein-metal ion, protein-protein, protein-DNA, protein-carbohydrate, carbohydrate-drug, peptide-peptide, DNA-drug and antigen-antibody. Approaches for the improvements of ACE in term of precision, rinsing protocols and sensitivity are discussed. The combined use of computer simulation programs to support data evaluation is presented. In conclusion, the performance of ACE is compared with other techniques such as equilibrium dialysis, parallel artificial membrane permeability assay, high-performance affinity chromatography as well as surface plasmon resonance, ultraviolet, circular dichroism, nuclear magnetic resonance, Fourier transform infrared, fluorescence, MS and isothermal titration calorimetry. PMID:25534793

  9. Interactions of Zn(II) Ions with Humic Acids Isolated from Various Type of Soils. Effect of pH, Zn Concentrations and Humic Acids Chemical Properties

    PubMed Central

    Boguta, Patrycja; Sokołowska, Zofia

    2016-01-01

    The main aim of this study was the analysis of the interaction between humic acids (HAs) from different soils and Zn(II) ions at wide concentration ranges and at two different pHs, 5 and 7, by using fluorescence and FTIR spectroscopy, as well as potentiometric measurements. The presence of a few areas of HAs structures responsible for Zn(II) complexing was revealed. Complexation at α-sites (low humified structures of low-molecular weight and aromatic polycondensation) and β-sites (weakly humified structures) was stronger at pH 7 than 5. This trend was not observed for γ-sites (structures with linearly-condensed aromatic rings, unsaturated bonds and large molecular weight). The amount of metal complexed at pH5 and 7 by α and γ-structures increased with a decrease in humification and aromaticity of HAs, contrary to β-areas where complexation increased with increasing content of carboxylic groups. The stability of complexes was higher at pH 7 and was the highest for γ-structures. At pH 5, stability decreased with C/N increase for α-areas and -COOH content increase for β-sites; stability increased with humification decrease for γ-structures. The stability of complexes at α and β-areas at pH 7 decreased with a drop in HAs humification. FTIR spectra at pH 5 revealed that the most-humified HAs tended to cause bidentate bridging coordination, while in the case of the least-humified HAs, Zn caused bidentate bridging coordination at low Zn additions and bidentate chelation at the highest Zn concentrations. Low Zn doses at pH 7 caused formation of unidentate complexes while higher Zn doses caused bidentate bridging. Such processes were noticed for HAs characterized by high oxidation degree and high oxygen functional group content; where these were low, HAs displayed bidentate bridging or even bidentate chelation. To summarize, the above studies have showed significant impact of Zn concentration, pH and some properties of HAs on complexation reactions of humic

  10. Determination of metal ions by fluorescence anisotropy exhibits a broad dynamic range

    NASA Astrophysics Data System (ADS)

    Thompson, Richard B.; Maliwal, Badri P.; Fierke, Carol A.

    1998-05-01

    Recently, we have shown that metal ions free in solution may be determined at low levels by fluorescence anisotropy (polarization) measurements. Anisotropy measurements enjoy the advantages of wavelength ratiometric techniques for determining metal ions such as calcium, because anisotropy measurements are ratiometric as well. Furthermore, fluorescence anisotropy may be imaged in the microscope. An advantage of anisotropy not demonstrated for wavelength ratiometric approaches using indicators such as Fura-2 and Indo-1 is that under favorable circumstances anisotropy-based determinations exhibit a much broader dynamic range in metal ion concentration. Determinations of free Zn(II) in the picomolar range are demonstrated.

  11. A new cyclic supramolecular Zn(II) complex derived from a N2O2 oxime chelate ligand with luminescence mechanochromism.

    PubMed

    Zhang, Shou-Ting; Li, Tian-Rong; Wang, Bao-Dui; Yang, Zheng-Yin; Liu, Jian; Wang, Zhi-Yi; Dong, Wen-Kui

    2014-02-21

    A new Zn(II) complex was synthesized based on a new Salen-type tetradentate N2O2 bisoxime chelate ligand (H2L) derived from 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP) and 1,2-bis(aminooxy)ethane. Single-crystal X-ray diffraction analysis reveals that the structure of the Zn(II) complex features a three-dimensional (3D) cyclic supramolecular system via intermolecular hydrogen bonds. Moreover, the solid-state photoluminescent properties demonstrate that the Zn(II) complex exhibits unusual luminescence mechanochromism tuned by CH3OH. PMID:24352216

  12. Harnessing a ratiometric fluorescence output from a sensor array.

    PubMed

    Wang, Zhuo; Palacios, Manuel A; Zyryanov, Grigory; Anzenbacher, Pavel

    2008-01-01

    Ratiometric fluorescence-based sensors are widely sought after because they can effectively convert even relatively small changes in optical output into a strong and easy-to-read signal. However, ratiometric sensor molecules are usually difficult to make. We present a proof-of-principle experiment that shows that efficient ratiometric sensing may be achieved by an array of two chromophores, one providing an on-to-off response and the second yielding an off-to-on response in a complementary fashion. In the case that both chromophores emit light of different color, the result is a switching of colors that may be utilized in the same way as from a true ratiometric probe. The chromophore array comprises two sensor elements: i) a polyurethane membrane with embedded N-anthracen-9-yl-methyl-N-7-nitrobenzoxa-[1,2,5]diazo-4-yl-N',N'-dimethylethylenediamine hydrochloride and ii) a membrane with N,N-dimethyl-N'-(9-methylanthracenyl)ethylenediamine. A combination of photoinduced electron transfer (PET) and fluorescence resonance energy transfer (FRET) allows for green-to-blue emission switching in the presence of Zn(II) ions. The sensing experiments carried out with different Zn(II) salts at controlled pH revealed that the degree of color switching in the individual sensor elements depends on both the presence of Zn(II) ions and the counter anion. These results suggest that sensing of both cations and anions may perhaps be extended to different cation-anion pairs. PMID:18688830

  13. Synthesis, structure and spectral properties of O,N,N coordinating ligands and their neutral Zn(ii) complexes: a combined experimental and theoretical study.

    PubMed

    Hens, Amar; Mondal, Pallab; Rajak, Kajal Krishna

    2013-10-01

    Mononuclear Zn(ii) complexes with the general formula [Zn(L)2] have been synthesized in good yields by reacting Zn(OAc)2 with HL in a ratio of 1 : 2 in methanol solvent. Here L is the deprotonated form of 6-[(quinolin-8-ylamino)methylene]cyclohexa-2,4-dienone (HL(1)), 4-chloro-6-[(quinolin-8-ylamino)methylene]cyclohexa-2,4-dienone (HL(2)), 4-methyl-6-[(quinolin-8-ylamino)methylene]cyclohexa-2,4-dienone (HL(3)), 2,4-dimethyl-6-[(quinolin-8-ylamino) methylene]cyclohexa-2,4-dienone (HL(4)), 2-methoxy-6-[(quinolin-8-ylamino) methylene]cyclohexa-2,4-dienone (HL(5)). The electronic structures and photophysical properties of the ligands were calculated by DFT and TDDFT methods. The X-ray structure of one complex is reported. The ligands have a strong binding ability [(0.75-15.37) × 10(4)] and ratiometric response to Zn(2+) ions. With the addition of Zn(2+) ions to the ligands in THF solution a sharp color change is observed visually, and as well a significant enhancement of the fluorescence intensity and the quantum yield for this series occurs. The introduction of other metal ions having biological and environmental effects results in either unaltered or quenched emission intensity. However we observed the sensing property of the ligands strongly depends on the substituent at the ortho position of the phenol group. DFT calculation reveals that the ICT process take place from the salicylaldehyde (donor moiety) to quinoline (acceptor moiety) which is responsible for the enhancement of the fluorescence intensity of ligands after complexation. PMID:23995072

  14. Phenoxy-bridged binuclear Zn(II) complex holding salen ligand: Synthesis and structural characterization

    NASA Astrophysics Data System (ADS)

    Azam, Mohammad; Al-Resayes, Saud I.

    2016-03-01

    A novel binuclear phenoxo-bridged zinc complex obtained from the interaction of ligand, 2,2-(1E,1E)-(2,2-dimethylpropane-1,3-diyl)bis(azanylylidene) bis(methanylylidene)diphenol with zinc chloride is reported. The synthesized and isolated zinc complex has been characterized by FT-IR, 1H- and 13C- NMR, ESI-MS, TGA/DTA and single crystal X-ray diffraction studies. The phenoxo-bridge in this binuclear Zn(II) complex is due to the phenolic oxygen of the salen liagnd. The complex crystallizes in monoclinic P-1 space group, and different geometry has been assigned for both zinc ions in the complex.

  15. Trinuclear Cage-Like Zn(II) Macrocyclic Complexes: Enantiomeric Recognition and Gas Adsorption Properties.

    PubMed

    Janczak, Jan; Prochowicz, Daniel; Lewiński, Janusz; Fairen-Jimenez, David; Bereta, Tomasz; Lisowski, Jerzy

    2016-01-11

    Three zinc(II) ions in combination with two units of enantiopure [3+3] triphenolic Schiff-base macrocycles 1, 2, 3, or 4 form cage-like chiral complexes. The formation of these complexes is accompanied by the enantioselective self-recognition of chiral macrocyclic units. The X-ray crystal structures of these trinuclear complexes show hollow metal-organic molecules. In some crystal forms, these barrel-shaped complexes are arranged in a window-to-window fashion, which results in the formation of 1D channels and a combination of both intrinsic and extrinsic porosity. The microporous nature of the [Zn3 12 ] complex is reflected in its N2 , Ar, H2 , and CO2 adsorption properties. The N2 and Ar adsorption isotherms show pressure-gating behavior, which is without precedent for any noncovalent porous material. A comparison of the structures of the [Zn3 12 ] and [Zn3 32 ] complexes with that of the free macrocycle H3 1 reveals a striking structural similarity. In H3 1, two macrocyclic units are stitched together by hydrogen bonds to form a cage very similar to that formed by two macrocyclic units stitched together by Zn(II) ions. This structural similarity is manifested also by the gas adsorption properties of the free H3 1 macrocycle. Recrystallization of [Zn3 12 ] in the presence of racemic 2-butanol resulted in the enantioselective binding of (S)-2-butanol inside the cage through the coordination to one of the Zn(II) ions. PMID:26642975

  16. Sol-gel encapsulation of binary Zn(II) compounds in silica nanoparticles. Structure-activity correlations in hybrid materials targeting Zn(II) antibacterial use.

    PubMed

    Halevas, E; Nday, C M; Kaprara, E; Psycharis, V; Raptopoulou, C P; Jackson, G E; Litsardakis, G; Salifoglou, A

    2015-10-01

    In the emerging issue of enhanced multi-resistant properties in infectious pathogens, new nanomaterials with optimally efficient antibacterial activity and lower toxicity than other species attract considerable research interest. In an effort to develop such efficient antibacterials, we a) synthesized acid-catalyzed silica-gel matrices, b) evaluated the suitability of these matrices as potential carrier materials for controlled release of ZnSO4 and a new Zn(II) binary complex with a suitably designed well-defined Schiff base, and c) investigated structural and textural properties of the nanomaterials. Physicochemical characterization of the (empty-loaded) silica-nanoparticles led to an optimized material configuration linked to the delivery of the encapsulated antibacterial zinc load. Entrapment and drug release studies showed the competence of hybrid nanoparticles with respect to the a) zinc loading capacity, b) congruence with zinc physicochemical attributes, and c) release profile of their zinc load. The material antimicrobial properties were demonstrated against Gram-positive (Staphylococcus aureus, Bacillus subtilis, Bacillus cereus) and negative (Escherichia coli, Pseudomonas aeruginosa, Xanthomonas campestris) bacteria using modified agar diffusion methods. ZnSO4 showed less extensive antimicrobial behavior compared to Zn(II)-Schiff, implying that the Zn(II)-bound ligand enhances zinc antimicrobial properties. All zinc-loaded nanoparticles were less antimicrobially active than zinc compounds alone, as encapsulation controls their release, thereby attenuating their antimicrobial activity. To this end, as the amount of loaded zinc increases, the antimicrobial behavior of the nano-agent improves. Collectively, for the first time, sol-gel zinc-loaded silica-nanoparticles were shown to exhibit well-defined antimicrobial activity, justifying due attention to further development of antibacterial nanotechnology. PMID:26198972

  17. Synthesis and characterization of VO(II), Co(II), Ni(II), Cu(II) and Zn(II) complexes of chromone based azo-linked Schiff base ligand

    NASA Astrophysics Data System (ADS)

    Anitha, C.; Sheela, C. D.; Tharmaraj, P.; Johnson Raja, S.

    2012-12-01

    Azo-Schiff-base complexes of VO(II), Co(II), Ni(II), Cu(II) and Zn(II) have been synthesized and characterized by elemental analysis, IR, UV-Vis, 1H NMR, mass spectra, molar conductance, magnetic susceptibility measurement, electron spin resonance (EPR), CV, fluorescence, NLO and SEM. The conductance data indicate the nonelectrolytic nature of the complexes, except VO(II) complex which is electrolytic in nature. On the basis of electronic spectra and magnetic susceptibility octahedral geometry has been proposed for the complexes. The EPR spectra of copper and oxovanadium complexes in DMSO at 300 and 77 K were recorded and its salient features are reported. The redox behavior of the copper(II) complex was studied using cyclic voltammetry. The in vitro antimicrobial activity against Staphylococcus aureus, Escherichia coli, Salmonella enterica typhi, Bacillus subtilis and Candida strains was studied and compared with that of free ligand by well-diffusion technique. The azo Schiff base exhibited fluorescence properties originating from intraligand (π-π∗) transitions and metal-mediated enhancement is observed on complexation and so the synthesized complexes can serve as potential photoactive materials as indicated from their characteristic fluorescence properties. On the basis of the optimized structures, the second-order nonlinear optical properties (NLO) are calculated by using second-harmonic generation (SHG) and also the surface morphology of the complexes was studied by SEM.

  18. Relationship between Ni(II) and Zn(II) Coordination and Nucleotide Binding by the Helicobacter pylori [NiFe]-Hydrogenase and Urease Maturation Factor HypB*

    PubMed Central

    Sydor, Andrew M.; Lebrette, Hugo; Ariyakumaran, Rishikesh; Cavazza, Christine; Zamble, Deborah B.

    2014-01-01

    The pathogen Helicobacter pylori requires two nickel-containing enzymes, urease and [NiFe]-hydrogenase, for efficient colonization of the human gastric mucosa. These enzymes possess complex metallocenters that are assembled by teams of proteins in multistep pathways. One essential accessory protein is the GTPase HypB, which is required for Ni(II) delivery to [NiFe]-hydrogenase and participates in urease maturation. Ni(II) or Zn(II) binding to a site embedded in the GTPase domain of HypB modulates the enzymatic activity, suggesting a mechanism of regulation. In this study, biochemical and structural analyses of H. pylori HypB (HpHypB) revealed an intricate link between nucleotide and metal binding. HpHypB nickel coordination, stoichiometry, and affinity were modulated by GTP and GDP, an effect not observed for zinc, and biochemical evidence suggests that His-107 coordination to nickel toggles on and off in a nucleotide-dependent manner. These results are consistent with the crystal structure of HpHypB loaded with Ni(II), GDP, and Pi, which reveals a nickel site distinct from that of zinc-loaded Methanocaldococcus jannaschii HypB as well as subtle changes to the protein structure. Furthermore, Cys-142, a metal ligand from the Switch II GTPase motif, was identified as a key component of the signal transduction between metal binding and the enzymatic activity. Finally, potassium accelerated the enzymatic activity of HpHypB but had no effect on the other biochemical properties of the protein. Altogether, this molecular level information about HpHypB provides insight into its cellular function and illuminates a possible mechanism of metal ion discrimination. PMID:24338018

  19. Characterization of sophorolipid biosurfactant produced by Cryptococcus sp. VITGBN2 and its application on Zn(II) removal from electroplating wastewater.

    PubMed

    Basak, Geetanjali; Das, Nilanjana

    2014-11-01

    The present study aimed at elucidating the role of biosurfactant produced by yeast for the removal of Zn(II) ions from electroplating wastewater. The yeast species isolated from CETP, Vellore, Tamilnadu was identified as Cryptococcus sp.VITGBN2, based on molecular techniques, and was found to be potent producer of biosurfactant in mineral salt media containing vegetable oil as additional carbon source. Chemical structure of the purified biosurfactant was identified as acidic diacetate sophorolipid through GC-MS analysis. Interaction of Zn(II) ions with biosurfactant was monitored using FT-IR, SEM and EDS analysis. Zn (II) removal at 100 mg l(-1) concentration was 84.8% compared were other synthetic surfactants (Tween 80 and sodium dodecyl sulphate), yeast mediated biosurfactant showed enhanced Zn (II) removal in batch mode. The role of biosurfactant on Zn(II) removal was evaluated in column mode packed with biosurfactant entrapped in sodium alginate beads. At a flow rate of 1 ml min(-1) and bed height of 12 cm, immobilized biosurfactant showed 94.34% Zn(II) removal from electroplating wastewater. The present study confirmed that Zn(II) removal was biosurfactant mediated. This is the first report establishing the involvement of yeast mediated biosurfactant in Zn(II) removal from wastewater. PMID:25522510

  20. pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-β peptide.

    PubMed

    Ghalebani, Leila; Wahlström, Anna; Danielsson, Jens; Wärmländer, Sebastian K T S; Gräslund, Astrid

    2012-05-11

    Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer's disease amyloid plaques. The amyloid-β (Aβ) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resolution NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with (15)N- and (13)C,(15)N-labeled Aβ(1-40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to Aβ may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the Aβ peptide under these conditions. PMID:22525674

  1. Batch and fixed-bed column studies for biosorption of Zn(II) ions onto pongamia oil cake (Pongamia pinnata) from biodiesel oil extraction.

    PubMed

    Shanmugaprakash, M; Sivakumar, V

    2015-12-01

    The present work, analyzes the potential of defatted pongamia oil cake (DPOC) for the biosorption of Zn(II) ions from aqueous solutions in the both batch and column mode. Batch experiments were conducted to evaluate the optimal pH, effect of adsorbent dosage, initial Zn(II) ions concentration and contact time. The biosorption equilibrium and kinetics data for Zn(II) ions onto the DPOC were studied in detail, using several models, among all it was found to be that, Freundlich and the second-order model explained the equilibrium data well. The calculated thermodynamic parameters had shown that the biosorption of Zn(II) ions was exothermic and spontaneous in nature. Batch desorption studies showed that the maximum Zn(II) recovery occurred, using 0.1 M EDTA. The Bed Depth Service Time (BDST) and the Thomas model was successfully employed to evaluate the model parameters in the column mode. The results indicated that the DPOC can be applied as an effective and eco-friendly biosorbent for the removal of Zn(II) ions in polluted wastewater. PMID:26366934

  2. Affinity Peptide for Targeted Detection of Dysplasia in Barrett's Esophagus

    PubMed Central

    Li, Meng; Anastassiades, Costas P.; Joshi, Bishnu; Komarck, Chris M.; Piraka, Cyrus; Elmunzer, Badih J.; Turgeon, Danielle K.; Johnson, Timothy D.; Appelman, Henry; Beer, David G.; Wang, Thomas D.

    2012-01-01

    Background & Aims Dysplasia is a pre-malignant condition in Barrett's esophagus that is difficult to detect on screening endoscopy because of its flat architecture and patchy distribution. Peptides are promising for use as novel molecular probes that identify cell surface targets unique to disease, and can be fluorescence-labeled for detection. We aim to select and validate an affinity peptide that binds to esophageal dysplasia for future clinical studies. Methods Peptide selection was performed using phage display by removing non-specific binders using Q-hTERT (intestinal metaplasia) cells and achieving specific binding against OE33 (esophageal adenocarcinoma) cells. Selective binding was confirmed on bound phage counts, ELISA, flow cytometry, competitive inhibition, and fluorescence microscopy. On stereomicroscopy, specific peptide binding to dysplasia on endoscopically resected specimens was assessed by rigorous registration of fluorescence intensity to histology in 1 mm intervals. Results The peptide sequence SNFYMPL was selected and demonstrated preferential binding to target cells on bound phage counts, ELISA, and flow cytometry. Reducing binding was observed on competition with unlabeled peptide in a dose dependent manner, an affinity of Kd = 164 nM was measured, and peptide binding to the surface of OE33 cells was validated on fluorescence microscopy. On esophageal specimens (n=12), the fluorescence intensity (mean±SEM) in 1 mm intervals classified histologically as squamous (n=145), intestinal metaplasia (n=83), dysplasia (n=61) and gastric mucosa (n=69) was 46.5±1.6, 62.3±5.8, 100.0±9.0, and 42.4±3.0 arb units, respectively. Conclusions The peptide sequence SNFYMPL binds specifically to dysplasia in Barrett's esophagus, and can be fluorescence-labeled to target pre-malignant mucosa on imaging. PMID:20637198

  3. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Sondek, John

    2004-09-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, and for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:18429272

  4. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Brill, Allison L; Pasker, Renee L

    2013-01-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, as well as for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:24510596

  5. Adsorption and desorption of Zn(II) and Cu(II) on Ca- alginate immobilized activated rice bran

    NASA Astrophysics Data System (ADS)

    Suratman, A.; Kamalia, N. Z.; Kusumawati, W. A.

    2016-02-01

    Ca-alginate immobilized activated rice bran has been used for adsorption of Zn(II) and Cu(II) from aqueous solution. The effect of the pH, kinetics model, adsorption isotherm and desorption on the adsorption performance was investigated. Activated rice bran was immobilized by the entrapment in alginate beads. The adsorption strength of Ca-alginate immobilized activated rice bran was compared to Ca-alginate and non-immobilized activated rice bran. The concentrations of adsorbed ions were analyzed using Atomic Absorption Spectrophotometer (AAS). The result showed that pH of 4.0 and the contact time of 120 min are the optimum condition for adsorption of Zn(II) and Cu(II). The adsorption kinetic of Zn(II) and Cu(II) followed the pseudo-second-order model with adsorption rate constant 4.9 x 10-2 and 3.14 g.mg-1.min-1, respectively. The both adsorption processes obeyed Langmuir isotherm with adsorption capacity of 2.03 and 2.42 mg.g-1 of adsorbent, respectively. The strength of Zn adsorption on Ca-alginate immobilized activated rice bran (86.63%) was more effective compared to Ca-alginate beads (60.96%) and activated rice bran (43.85%). The strength of Cu adsorption was 80.00%, 61.50% and 22.10%, respectively. The desorption of Zn(II) and Cu(II) showed that recovery percentage of the adsorption was 76.56% and 57.80% with the condition of using HCl 0.1 M as desorption agent for 1 hour.

  6. Adsorptive removal of Zn(II) ion from aqueous solution using rice husk-based activated carbon

    NASA Astrophysics Data System (ADS)

    Taha, Mohd F.; Ibrahim, Muhammad H. C.; Shaharun, Maizatul S.; Chong, F. K.

    2012-09-01

    The study of rice husk-based activated carbon as a potential low-cost adsorbent for the removal of Zn(II) ion from aqueous solution was investigated. Rice husk, an agricultural waste, is a good alternative source for cheap precursor of activated carbon due to its abundance and constant availability. In this work, rice husk-based activated carbon was prepared via chemical treatment using NaOH as an activation agent prior the carbonization process. Three samples, i.e. raw rice husk, rice husk treated with NaOH and rice husk-based activated carbon carbonized at 650°C, were analyzed for their morphological characteristics using field-emission scanning electron microscope/energy dispersive X-ray (FESEM/EDX). Other analyses were also conducted on these samples using fourier transmitter infrared spectroscopy (FTIR), CHN elemental analyzer and X-ray diffraction (XRD) for characterization study. The porous properties of rice husk-based activated carbon were determined by Brunauer-Emmett-Teller (BET) surface area analyzer, and its surface area and pore volume were found to be 255 m2/g and 0.17 cm2/g, respectively. The adsorption studies for the removal of Zn(II) ion from aqueous solution were carried out as a function of varied contact time at room temperature. The concentration of Zn(II) ion was analyzed using atomic absorption spectrophotometer (AAS). The results obtained from adsorption studies indicate the potential of rice husk as an economically promising precursor for the preparation of activated carbon for removal of Zn(II) ion from aqueous solution.

  7. Synthesis, magnetism and spectral studies of six defective dicubane tetranuclear {M4O6} (M = Ni(II), Co(II), Zn(II)) and three trinuclear Cd(II) complexes with polydentate Schiff base ligands.

    PubMed

    Jiang, Lin; Zhang, Dong-Yan; Suo, Jing-Jing; Gu, Wen; Tian, Jin-Lei; Liu, Xin; Yan, Shi-Ping

    2016-06-21

    A series of Ni(II), Co(II), Zn(II) and Cd(II) complexes with polytopic Schiff base ligands have been synthesized. The single-crystal X-ray crystallography results show that tetranuclear complexes have common face-shared defective dicubane cores, whereas trinuclear Cd(II) complexes are almost linear entities. Synthesis methods (solvent evaporation and hydrothermal synthesis), reaction conditions (pH, solvents and dosage) and coligands (azide, methanol, chloride and acetate) play vital roles in determining the final structure of the complexes and therefore their magnetic properties. In complexes , the terminal and central M(2+) ions are connected through mixed bridges, μ-phenoxido/μ1,1,1-X and μ-Oalphatic/μ1,1,1-X, while central two M(2+) ions are linked by double bridges, μ1,1,1-X (X = azido and methoxido groups for and respectively). For complex , two central Ni(II) ions are connected through two μ1,1,1-N3(-) which is relatively less reported. For complexes , there are two kinds of Cd(II), the centre Cd(II) ions are eight-coordinated with triangle dodecahedral geometries, while the two side Cd(II) ions are six-coordinated with trigonal prism geometries using chlorides or acetates as terminal ligands. Magnetic susceptibility measurements (χM) for compounds have been performed, and they reveal predominant ferromagnetic exchange interactions in Co(II) and Ni(II) tetramers. The photoluminescence studies show that the Zn(II) complex and three Cd(II) complexes have strong fluorescence, and the lifetimes are measured to be in the 10(2) nanosecond timescale. PMID:27230103

  8. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  9. Quantifying Affinity among Chinese Dialects.

    ERIC Educational Resources Information Center

    Cheng, Chin-Chuan

    A study of the relationships between Chinese dialects based on a quantitative measure of dialect affinity is summarized. First, tone values in all the dialect localities available in the early 1970s were used to calculate the dialectal differences in terms of tone height with respect to the "yin and yang" split. In the late 1970s, calculations of…

  10. Theoretical studies on the first proton macroaffinity of Ni(II), Cu(II), Zn(II) and Cd(II) complexes of four triazacycloalkanes ([X]ane N3, X = 9-12): good correlations with the formation constants in solution.

    PubMed

    Salehzadeh, Sadegh; Shooshtari, Amir; Bayat, Mehdi

    2009-04-21

    A theoretical study on the first protonation step of Ni(ii), Cu(ii), Zn(ii) and Cd(ii) complexes of some triazacycloalkanes with general formula [X]ane N(3) (X = 9-12) is reported. The calculations were performed at DFT (B3LYP) level of theory, using LanL2DZ basis set. The DFT calculations were performed again using DZVP2 basis set for Ni(ii), Cu(ii) and Zn(ii) complexes and DZVP for Cd(ii) complexes. Once again, two kinds of our recently published definitions for gas-phase proton affinities of polybasic ligands, proton microaffinity and proton macroaffinity, were extended to their metal complexes. Among the 16 investigated complexes the most stable complex has both the smallest proton macroaffinity and macrobasicity. The least stable complex has also both the greatest proton macroaffinity and macrobasicity. In the case of each metal ion there are good correlations between the calculated gas-phase proton macroaffinities as well as macrobasicities of the corresponding complexes with their formation constants in solution. PMID:19333512

  11. Reaction-based fluorescent sensor for investigating mobile Zn2+ in mitochondria of healthy versus cancerous prostate cells

    PubMed Central

    Chyan, Wen; Zhang, Daniel Y.; Lippard, Stephen J.; Radford, Robert J.

    2014-01-01

    Chelatable, mobile forms of divalent zinc, Zn(II), play essential signaling roles in mammalian biology. A complex network of zinc import and transport proteins has evolved to control zinc concentration and distribution on a subcellular level. Understanding the action of mobile zinc requires tools that can detect changes in Zn(II) concentrations at discrete cellular locales. We present here a zinc-responsive, reaction-based, targetable probe based on the diacetyled form of Zinpyr-1. The compound, (6-amidoethyl)triphenylphosphonium Zinpyr-1 diacetate (DA-ZP1-TPP), is essentially nonfluorescent in the metal-free state; however, exposure to Zn(II) triggers metal-mediated hydrolysis of the acetyl groups to afford a large, rapid, and zinc-induced fluorescence response. DA-ZP1-TPP is insensitive to intracellular esterases over a 2-h period and is impervious to proton-induced turn-on. A TPP unit is appended for targeting mitochondria, as demonstrated by live cell fluorescence imaging studies. The practical utility of DA-ZP1-TPP is demonstrated by experiments revealing that, in contrast to healthy epithelial prostate cells, tumorigenic cells are unable to accumulate mobile zinc within their mitochondria. PMID:24335702

  12. Synthesis and luminescence properties of polymeric complexes of Cu(II), Zn(II) and Al(III) with 8-hydroxyquinoline side group-containing polystyrene

    NASA Astrophysics Data System (ADS)

    Gao, Baojiao; Wei, Xiaopeng; Zhang, Yanyan

    2013-01-01

    Three kinds of metalloquinolate-containing polystyrene were prepared via a polymer reaction and a coordination reaction. 5-Chloromethyl-8-hydroxyquinoline (CHQ) was first prepared through the chloromethylation reaction of 8-hydroxyquinoline (HQ) with 1,4-bichloromethoxy-butane as chloromethylation reagent. A polymer reaction, Friedel-Crafts alkylation reaction, was carried out between polystyrene (PS) and CHQ in the presence of Lewis catalyst, and HQ was bonded onto the side chains of PS, obtaining 8-hydroxyquinoline-functionalized Polystyrene, HQ-PS. And then, by using one-pot method with two-stage procedures, the coordination reaction of HQ-PS and small molecule HQ with metal ions including Al(III), Zn(II) and Cu(II) ions, was allowed to be carried out, and three polymeric metalloquinolates, AlQ3-PS, ZnQ2-PS and CuQ2-PS, were successfully prepared, respectively. In the chemical structures of these polymeric metalloquinolates, metalloquinolates were chemically attached onto the side chains of PS. HQ-PS and three polymeric metalloquinolates were fully characterized by FTIR, 1H NMR and TGA. The luminescence properties of the three polymeric metalloquinolates were mainly investigated by UV/Vis absorption spectra and fluorescence emission spectra in solutions and in solid film states. When excited by the ray at about 365 nm, the three polymeric metalloquinolates have blue-green luminescence, and the main emission peaks in the DMF solutions are located at 490, 482 and 502 nm for AlQ3-PS, ZnQ2-PS and CuQ2-PS, respectively. As compared with their emissions in solutions, the emissions in solid film states are red-shifted to some extent, and the main emission peaks are located at 500, 488 and 510 nm for AlQ3-PS, ZnQ2-PS and CuQ2-PS, respectively. Besides, these polymeric metalloquinolates have higher thermal stability than PS as polymeric skeleton.

  13. Interaction with biomacromolecules and antiproliferative activities of Mn(II), Ni(II), Zn(II) complexes of demethylcantharate and 2,2'-bipyridine.

    PubMed

    Zhang, Fan; Lin, Qiu-Yue; Hu, Wan-Li; Song, Wen-Ji; Shen, Shu-Ting; Gui, Pan

    2013-06-01

    Three new transition metal complexes [Mn2(DCA)2(bipy)2]·5H2O (1), [M2(DCA)2(bipy)2(H2O)]·10H2O(M=Ni(II)(2);Zn(II)(3)), (DCA=demethylcantharate, 7-oxabicyclo[2,2,1]heptane-2,3-dicarboxylate, C8H8O5) were synthesized and characterized by elemental analysis, molar conductance, infrared spectra and X-ray diffraction techniques. Each metal ion was six-coordinated in complexes. Complex 1 has a Mn2O2 center. Complexes 2 and 3 have asymmetric binuclear structure. Great amount of intermolecular hydrogen-bonding and π-π(*) stacking interactions were formed in these complex structures. The DNA-binding properties of complexes were investigated by electronic absorption spectra and viscosity measurements. The DNA binding constants Kb/(Lmol(-1)) were 1.71×10(4) (1), 2.62×10(4) (2) and 1.59×10(4) (3) at 298 K. The complexes could quench the intrinsic fluorescence of bovine serum albumin (BSA) strongly through static quenching. The protein binding constants Ka/(L mol(-1)) were 7.27×10(4) (1), 4.55×10(4) (2) and 7.87×10(4) L mol(-1) (3) and binding site was one. The complexes bind more tightly with DNA and BSA than with ligands. Complexes 1 and 3 had stronger inhibition ratios than Na2(DCA) against human hepatoma cells (SMMC-7721) lines and human gastric cancer cells (MGC80-3) lines in vitro. Complex 3 showed the strongest antiproliferative activity against SMMC-7721 (IC50=29.46±2.12 μmol L(-1)) and MGC80-3 (IC50=27.02±2.38 μmol L(-1)), which shows potential in anti-cancer drug development. PMID:23557779

  14. Interaction with biomacromolecules and antiproliferative activities of Mn(II), Ni(II), Zn(II) complexes of demethylcantharate and 2,2'-bipyridine

    NASA Astrophysics Data System (ADS)

    Zhang, Fan; Lin, Qiu-Yue; Hu, Wan-Li; Song, Wen-Ji; Shen, Shu-Ting; Gui, Pan

    2013-06-01

    Three new transition metal complexes [Mn2(DCA)2(bipy)2]·5H2O (1), [M2(DCA)2(bipy)2(H2O)]·10H2O(M = Ni(II)(2);Zn(II)(3)), (DCA = demethylcantharate, 7-oxabicyclo[2,2,1]heptane-2,3-dicarboxylate, C8H8O5) were synthesized and characterized by elemental analysis, molar conductance, infrared spectra and X-ray diffraction techniques. Each metal ion was six-coordinated in complexes. Complex 1 has a Mn2O2 center. Complexes 2 and 3 have asymmetric binuclear structure. Great amount of intermolecular hydrogen-bonding and π-π* stacking interactions were formed in these complex structures. The DNA-binding properties of complexes were investigated by electronic absorption spectra and viscosity measurements. The DNA binding constants Kb/(L mol-1) were 1.71 × 104 (1), 2.62 × 104 (2) and 1.59 × 104 (3) at 298 K. The complexes could quench the intrinsic fluorescence of bovine serum albumin (BSA) strongly through static quenching. The protein binding constants Ka/(L mol-1) were 7.27 × 104 (1), 4.55 × 104 (2) and 7.87 × 104 L mol-1 (3) and binding site was one. The complexes bind more tightly with DNA and BSA than with ligands. Complexes 1 and 3 had stronger inhibition ratios than Na2(DCA) against human hepatoma cells (SMMC-7721) lines and human gastric cancer cells (MGC80-3) lines in vitro. Complex 3 showed the strongest antiproliferative activity against SMMC-7721 (IC50 = 29.46 ± 2.12 μmol L-1) and MGC80-3 (IC50 = 27.02 ± 2.38 μmol L-1), which shows potential in anti-cancer drug development.

  15. The meloxicam complexes of Co(II) and Zn(II): Synthesis, crystal structures, photocleavage and in vitro DNA-binding

    NASA Astrophysics Data System (ADS)

    Sanatkar, Tahereh Hosseinzadeh; Hadadzadeh, Hassan; Simpson, Jim; Jannesari, Zahra

    2013-10-01

    Two neutral mononuclear complexes of Co(II) and Zn(II) with the non-steroidal anti-inflammatory drug meloxicam (H2mel, 4-hydroxy-2-methyl-N-(5-methyl-2-thiazolyl)-2H-1,2-benzothiazine-3-carboxammide-1,1-dioxide), [Co(Hmel)2(EtOH)2] (1), and [Zn(Hmel)2(EtOH)2] (2), were synthesized and characterized by elemental analysis, IR and UV-Vis spectroscopy and their solid-state structures were studied by single-crystal diffraction. The complexes have a distorted octahedral geometry around the metal atom. The experimental data indicate that the meloxicam acts as a deprotonated bidentate ligand (through the amide oxygen and the nitrogen atom of the thiazolyl ring) in the complexes, and a strong intramolecular hydrogen bond between the amide N-H function and the enolate O atom stabilizes the ZZZ conformation of meloxicam ligands. Absorption, fluorescence spectroscopy and cyclic voltammetry have been used to investigate the binding of the complexes with fish sperm DNA (FS-DNA). Additionally, the photocleavage studies have been also used to investigate the binding of the complexes with plasmid DNA. The interaction of the complexes with DNA was monitored by a blue shift and hyperchromism in the UV-Vis spectra attributed to an electrostatic binding mode. A competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The experimental results show that the complexes can cleave pUC57 plasmid DNA.

  16. Comparative Study on the Efficiency of the Photodynamic Inactivation of Candida albicans Using CdTe Quantum Dots, Zn(II) Porphyrin and Their Conjugates as Photosensitizers.

    PubMed

    Viana, Osnir S; Ribeiro, Martha S; Rodas, Andréa C D; Rebouças, Júlio S; Fontes, Adriana; Santos, Beate S

    2015-01-01

    The application of fluorescent II-VI semiconductor quantum dots (QDs) as active photosensitizers in photodymanic inactivation (PDI) is still being evaluated. In the present study, we prepared 3 nm size CdTe QDs coated with mercaptosuccinic acid and conjugated them electrostatically with Zn(II) meso-tetrakis (N-ethyl-2-pyridinium-2-yl) porphyrin (ZnTE-2-PyP or ZnP), thus producing QDs-ZnP conjugates. We evaluated the capability of the systems, bare QDs and conjugates, to produce reactive oxygen species (ROS) and applied them in photodynamic inactivation in cultures of Candida albicans by irradiating the QDs and testing the hypothesis of a possible combined contribution of the PDI action. Tests of in vitro cytotoxicity and phototoxicity in fibroblasts were also performed in the presence and absence of light irradiation. The overall results showed an efficient ROS production for all tested systems and a low cytotoxicity (cell viability >90%) in the absence of radiation. Fibroblasts incubated with the QDs-ZnP and subjected to irradiation showed a higher cytotoxicity (cell viability <90%) depending on QD concentration compared to the bare groups. The PDI effects of bare CdTe QD on Candida albicans demonstrated a lower reduction of the cell viability (~1 log10) compared to bare ZnP which showed a high microbicidal activity (~3 log10) when photoactivated. The QD-ZnP conjugates also showed reduced photodynamic activity against C. albicans compared to bare ZnP and we suggest that the conjugation with QDs prevents the transmembrane cellular uptake of the ZnP molecules, reducing their photoactivity. PMID:25993419

  17. Theoretical proton affinity and fluoride affinity of nerve agent VX.

    PubMed

    Bera, Narayan C; Maeda, Satoshi; Morokuma, Keiji; Viggiano, Al A

    2010-12-23

    Proton affinity and fluoride affinity of nerve agent VX at all of its possible sites were calculated at the RI-MP2/cc-pVTZ//B3LYP/6-31G* and RI-MP2/aug-cc-pVTZ//B3LYP/6-31+G* levels, respectively. The protonation leads to various unique structures, with H(+) attached to oxygen, nitrogen, and sulfur atoms; among which the nitrogen site possesses the highest proton affinity of -ΔE ∼ 251 kcal/mol, suggesting that this is likely to be the major product. In addition some H(2), CH(4) dissociation as well as destruction channels have been found, among which the CH(4) + [Et-O-P(═O)(Me)-S-(CH(2))(2)-N(+)(iPr)═CHMe] product and the destruction product forming Et-O-P(═O)(Me)-SMe + CH(2)═N(+)(iPr)(2) are only 9 kcal/mol less stable than the most stable N-protonated product. For fluoridization, the S-P destruction channel to give Et-O-P(═O)(Me)(F) + [S-(CH(2))(2)-N-(iPr)(2)](-) is energetically the most favorable, with a fluoride affinity of -ΔE ∼ 44 kcal. Various F(-) ion-molecule complexes are also found, with the one having F(-) interacting with two hydrogen atoms in different alkyl groups to be only 9 kcal/mol higher than the above destruction product. These results suggest VX behaves quite differently from surrogate systems. PMID:21117653

  18. Electrochemical affinity biosensors for detection of mycotoxins: A review.

    PubMed

    Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

    2013-11-15

    This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago. PMID:23743326

  19. Synthesis and spectral characterization of Zn(II) microsphere series for antimicrobial application

    NASA Astrophysics Data System (ADS)

    Singh, Ajay K.; Pandey, Sarvesh K.; Pandey, O. P.; Sengupta, S. K.

    2014-09-01

    Microsphere series have been synthesized by reacting zinc(II) acetate dihydrate with Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thiadiazole/oxadiazole/triazole with salicylaldehyde. Elemental analysis suggests that the complexes have 1:2 and 1:1 stoichiometry of the type [Zn(L)2(H2O)2] and [Zn(L‧)(H2O)2]; LH = Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thia/oxadiazole with salicylaldehyde; L‧H2 = Schiff bases derived from 3-(substituted phenyl)-4-amino-5-hydrazino-1,2,4-triazole and salicylaldehyde and were characterized by elemental analyses, IR, 1H NMR and 13C NMR spectral data. Scanning electron microscopy (SEM) showed that synthesized materials have microsphere like structure and there EDX analysis comparably matches with elemental analysis. For the antimicrobial application Schiff bases and their zinc(II) complexes were screened for four bacteria e.g. Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhi, Streptococcus pyogenes and four fungi e.g. Cyrtomium falcatum, Aspergillus niger, Fusarium oxysporium and Curvularia pallescence by the reported method. Schiff base and Zn(II) compounds showed significant antimicrobial activities. However, activities increase upon chelation. Thermal analysis (TGA) data of compound (10) showed its stability up to 300 °C.

  20. Two new luminescent Zn(II) compounds constructed from guanazole and aromatic polycarboxylate ligands

    NASA Astrophysics Data System (ADS)

    Zhao, Haixiang; Dong, Yanli; Liu, Haiping

    2016-02-01

    Two new Zn(II) compounds, namely [(CH3)2NH2]2n[Zn3(bpt)2(datrz)2]n (1) and [(CH3)2NH2)]n[Zn2(bptc)(datrz)]n·n(H2O) (2) (H3bpt = biphenyl-3,4‧,5-tricarboxylic acid, H4bptc = biphenyl-3,3‧,5,5‧-tetracarboxylic acid, Hdatrz = 3,5-diamino-1,2,4-triazole), have been obtained by the self-assemble reactions of Zn(NO3)2, 3,5-diamino-1,2,4-triazole, aromatic polycarboxylate ligands under solvothermal conditions. Single crystal X-ray structural analyses reveal that both compounds display three-dimensional (3D) frameworks. Compound 1 features a trinodal (3, 4, 6)-connected topological network with the point symbol of {4.62}2{4.64.8}{46.64.85}. Compound 2 displays a binodal (4, 6)-connected topological network with the point symbol of {32.62.72}{34.42.64.75}. In addition, the thermal stabilities and luminescent properties of compounds 1 and 2 were also investigated in the solid state at room temperature.

  1. Fluorescence Analysis of Sulfonamide Binding to Carbonic Anhydrase

    ERIC Educational Resources Information Center

    Wang, Sheila C.; Zamble, Deborah B.

    2006-01-01

    A practical laboratory experiment is described that illustrates the application of fluorescence resonance energy transfer to the study of protein-ligand binding. The affinities of wild-type and mutant human carbonic anhydrase II for dansylamide were determined by monitoring the increase in ligand fluorescence that occurs due to energy transfer…

  2. Bioinspired fluorescent dipeptide nanoparticles for targeted cancer cell imaging and real-time monitoring of drug release

    NASA Astrophysics Data System (ADS)

    Fan, Zhen; Sun, Leming; Huang, Yujian; Wang, Yongzhong; Zhang, Mingjun

    2016-04-01

    Peptide nanostructures are biodegradable and are suitable for many biomedical applications. However, to be useful imaging probes, the limited intrinsic optical properties of peptides must be overcome. Here we show the formation of tryptophan–phenylalanine dipeptide nanoparticles (DNPs) that can shift the peptide's intrinsic fluorescent signal from the ultraviolet to the visible range. The visible emission signal allows the DNPs to act as imaging and sensing probes. The peptide design is inspired by the red shift seen in the yellow fluorescent protein that results from π–π stacking and by the enhanced fluorescence intensity seen in the green fluorescent protein mutant, BFPms1, which results from the structure rigidification by Zn(II). We show that DNPs are photostable, biocompatible and have a narrow emission bandwidth and visible fluorescence properties. DNPs functionalized with the MUC1 aptamer and doxorubicin can target cancer cells and can be used to image and monitor drug release in real time.

  3. Lectin affinity chromatography of glycolipids

    SciTech Connect

    Torres, B.V.; Smith, D.F.

    1987-05-01

    Since glycolipids (GLs) are either insoluble or form mixed micelles in water, lectin affinity chromatography in aqueous systems has not been applied to their separation. They have overcome this problem by using tetrahydrofuran (THF) in the mobile phase during chromatography. Affinity columns prepared with the GalNAc-specific Helix pomatia agglutinin (HPA) and equilibrated in THF specifically bind the (/sup 3/H)oligosaccharide derived from Forssman GL indicating that the immobilized HPA retained its carbohydrate-binding specificity in this solvent. Intact Forssman GL was bound by the HPA-column equilibrated in THF and was specifically eluted with 0.1 mg/ml GalNAc in THF. Purification of the Forssman GL was achieved when a crude lipid extract of sheep erythrocyte membranes was applied to the HPA-column in THF. Non-specifically bound GLs were eluted from the column using a step gradient of aqueous buffer in THF, while the addition of GalNAc was required to elute the specifically bound GLs. Using this procedure the A-active GLs were purified from a crude lipid extract of type A human erythrocytes in a single chromatographic step. The use of solvents that maintain carbohydrate-binding specificity and lipid solubility will permit the application of affinity chromatography on immobilized carbohydrate-binding proteins to intact GLs.

  4. pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-{beta} peptide

    SciTech Connect

    Ghalebani, Leila; Wahlstroem, Anna; Danielsson, Jens; Waermlaender, Sebastian K.T.S.; Graeslund, Astrid

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Cu(II) and Zn(II) display pH-dependent binding to the A{beta}(1-40) peptide. Black-Right-Pointing-Pointer At pH 7.4 both metal ions display residue-specific binding to the A{beta} peptide. Black-Right-Pointing-Pointer At pH 5.5 the binding specificity is lost for Zn(II). Black-Right-Pointing-Pointer Differential Cu(II) and Zn(II) binding may help explain metal-induced AD toxicity. -- Abstract: Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer's disease amyloid plaques. The amyloid-{beta} (A{beta}) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resolution NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with {sup 15}N- and {sup 13}C,{sup 15}N-labeled A{beta}(1-40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to A{beta} may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the A{beta} peptide under these conditions.

  5. Fluorescent refrigeration

    DOEpatents

    Epstein, Richard I.; Edwards, Bradley C.; Buchwald, Melvin I.; Gosnell, Timothy R.

    1995-01-01

    Fluorescent refrigeration is based on selective radiative pumping, using substantially monochromatic radiation, of quantum excitations which are then endothermically redistributed to higher energies. Ultimately, the populated energy levels radiatively deexcite emitting, on the average, more radiant energy than was initially absorbed. The material utilized to accomplish the cooling must have dimensions such that the exciting radiation is strongly absorbed, but the fluorescence may exit the material through a significantly smaller optical pathlength. Optical fibers and mirrored glasses and crystals provide this requirement.

  6. Fluorous-assisted metal chelate affinity extraction technique for analysis of protein kinase activity.

    PubMed

    Hayama, Tadashi; Kiyokawa, Ena; Yoshida, Hideyuki; Imakyure, Osamu; Yamaguchi, Masatoshi; Nohta, Hitoshi

    2016-08-15

    We have developed a fluorous affinity-based extraction method for measurement of protein kinase activity. In this method, a fluorescent peptide substrate was phosphorylated by a protein kinase, and the obtained phosphopeptide was selectively captured with Fe(III)-immobilized perfluoroalkyliminodiacetic acid reagent via a metal chelate affinity technique. Next, the captured phosphopeptide was selectively extracted into a fluorous solvent mixture, tetradecafluorohexane and 1H,1H,2H,2H-tridecafluoro-1-n-octanol (3:1, v/v), using the specificity of fluorous affinity (fluorophilicity). In contrast, the remained substrate peptide in the aqueous (non-fluorous) phase was easily measured fluorimetrically. Finally, the enzyme activity could be assayed by measuring the decrease in fluorescence. The feasibility of this method was demonstrated by applying the method for measurement of the activity of cAMP-dependent protein kinase (PKA) using its substrate peptide (kemptide) pre-labeled with carboxytetramethylrhodamine (TAMRA). PMID:27260427

  7. Potential application of sludge produced from coal mine drainage treatment for removing Zn(II) in an aqueous phase.

    PubMed

    Cui, Mingcan; Jang, Min; Cho, Sang-Hyun; Khim, Jeehyeong

    2011-01-01

    Various analyses of physico-chemical characteristics and batch tests were conducted with the sludge obtained from a full-scale electrolysis facility for treating coal mine drainage in order to find the applicability of sludge as a material for removing Zn(II) in an aqueous phase. The physico-chemical analysis results indicated that coal mine drainage sludge (CMDS) had a high specific surface area and also satisfied the standard of toxicity characteristic leaching procedure (TCLP) because the extracted concentrations of certain toxic elements such as Pb, Cu, As, Hg, Zn, and Ni were much less than their regulatory limits. The results of X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) showed that the CMDS mainly consists of goethite (70%) and calcite (30%) as a weight basis. However, the zeta potential analysis represented that the CMDS had a lower isoelectric point of pH (pH(IEP)) than that of goethite or calcite. This might have been caused by the complexation of negatively charged anions, especially sulfate, which usually exists with a high concentration in coal mine drainage. The results of Fourier transform infrared (FT-IR) spectrometry analysis revealed that Zn(II) was dominantly removed as a form of precipitation by calcite, such as smithsonite [ZnCO₃] or hydrozincite [Zn₅(CO₃)₂(OH)₆]. Recycling sludge, originally a waste material, for the removal process of Zn(II), as well as other heavy metals, could be beneficial due to its high and speedy removal capability and low economic costs. PMID:21063752

  8. Structural analysis of complexes formed by ethyl 4-phenylthiocarbamoyl piperazine-1-carboxylate with Ni(II), Zn(II) and Cd(II) through spectroscopic and DFT techniques

    NASA Astrophysics Data System (ADS)

    Prakash, Om; Gautam, Priyanka; Dani, R. K.; Nandi, Abhisikta; Singh, N. K.; Singh, Ranjan K.

    2014-04-01

    A piperazine derivative, ethyl 4-phenylthiocarbamoyl piperazine-1-carboxylate and its Ni(II), Zn(II) and Cd(II) complexes have been synthesized and characterized by elemental analyses, magnetic susceptibility measurement, UV-Visible, FTIR, Raman spectroscopic and DFT methods. The Ni(II) and Zn(II) bind through the N and S sites of the two ligand Heptpc and N site of two pyridine molecules. However, the Cd(II) binds through the only N sites of the two ligand Heptpc and N site of two pyridine molecules. On the basis of various techniques used for the characterizations of the complexes, we found that the most possible geometry of the Ni(II) and Zn(II) complexes are distorted octahedral and of the Cd(II) complex is distorted tetrahedral.

  9. PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

    PubMed Central

    Walkup, Ward G.; Kennedy, Mary B.

    2014-01-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ~ 90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. PMID:24607360

  10. Immobilized metal ion affinity chromatography.

    PubMed

    Yip, T T; Hutchens, T W

    1992-01-01

    Immobilized metal ion affinity chromatography (IMAC) (1,2) is also referred to as metal chelate chromatography, metal ion interaction chromatography, and ligand-exchange chromatography. We view this affinity separation technique as an intermediate between highly specific, high-affinity bioaffinity separation methods, and wider spectrum, low-specificity adsorption methods, such as ion exchange. The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups (e.g., His residues) in peptides (e.g., 3-7) and on protein surfaces (8-13). The number of stationary phases that can be synthesized for efficient chelation of metal ions is unlimited, but the critical consideration is that there must be enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. Several examples are presented in Fig. 1. The challenge to produce new immobilized chelating groups, including protein surface metal-binding domains (14,15) is being explored continuously. Table 1 presents a list of published procedures for the synthesis and use of stationary phases with immobilized chelating groups. This is by no means exhaustive, and is intended only to give an idea of the scope and versatility of IMAC. Fig. 1 Schematic illustration of several types of immobilized metal-chelating groups, including, iminodiacetate (IDA), tris(carboxymethyl) ethylenediamine (TED), and the metal-binding peptides (GHHPH)(n)G (where n = 1,2,3, and 5) (14,15). Table 1 Immobilized Chelating Groups and Metal Ions Used for Immobilized Metal Ion Affinity Chromatography Chelating group Suitable metal ions Reference Commercial source Immodiacetate Transitional1,2 Pharmacia LKB Pierce Sigma Boehringer Mannheim TosoHaas 2-Hydroxy-3[N-(2- pyrtdylmethyl) glycme]propyl Transitional3 Not available ?-Alky1 mtrilo triacetic acid Transitional4 Not available Carboxymethylated asparhc acid Ca(II)13 Not available Tris (carboxy- methyl) ethylene Diamme

  11. Computational molecular characterization of the flavonoid Morin and its Pt(II), Pd(II) and Zn(II) complexes.

    PubMed

    Payán-Gómez, Sergio A; Flores-Holguín, Norma; Pérez-Hernández, Antonino; Piñón-Miramontes, Manuel; Glossman-Mitnik, Daniel

    2011-05-01

    In this work, we make use of a model chemistry within density functional theory (DFT) recently presented, which is called M05-2X, to calculate the molecular structure of the flavonoid Morin and its Pt(II), Pd(II) and Zn(II) complexes, as well to predict their IR and UV-Vis spectra, the dipole moment and polarizability, the free energy of solvation in different solvents as an indication of solubility, the HOMO and LUMO orbitals, and the chemical reactivity parameters that arise from Conceptual DFT. The calculated values are compared with the available experimental data for these molecules. PMID:20628776

  12. Biosorption of Cr(VI) and Zn(II) ions from aqueous solution onto the solid biodiesel waste residue: mechanistic, kinetic and thermodynamic studies.

    PubMed

    Muthusamy, Shanmugaprakash; Venkatachalam, Sivakumar; Jeevamani, Prasana Manikanda Kartick; Rajarathinam, Nandusha

    2014-01-01

    In this present study, the biosorption of Cr(VI) and Zn(II) ions from synthetic aqueous solution on defatted J atropha oil cake (DJOC) was investigated. The effect of various process parameters such as the initial pH, adsorbent dosage, initial metal ion concentration and contact time has been studied in batch-stirred experiments. Maximum removal of Cr(VI) and Zn(II) ions in aqueous solution was observed at pH 2.0 and pH. 5.0, respectively. The removal efficiency of Cr(VI) and Zn(II) ions from the aqueous solution was found to be 72.56 and 79.81%, respectively, for initial metal ion concentration of 500 mg/L at 6 g/L dosage concentration. The biosorbent was characterized by Fourier transform infrared, scanning electron microscopy and zero point charge. Equilibrium data were fitted to the Langmuir, Freundlich, Temkin and Dubinin-Radushkevich isotherm models and the best fit is found to be with the Freundlich isotherm for both Cr(VI) and Zn(II) metal ions. The kinetic data obtained at different metal ion concentration have been analysed using the pseudo-first-order, pseudo-second-order and intraparticle diffusion models and were found to follow the pseudo-second-order kinetic model. The values of mass transfer diffusion coefficients (De) were determined by Boyd model and compared with literature values. Various thermodynamic parameters, such as ΔG°, ΔH° and ΔS°, were analysed using the equilibrium constant values (Ke) obtained from experimental data at different temperatures. The results showed that biosorption of Cr(VI) and Zn(II) ions onto the DJOC system is more spontaneous and exothermic in nature. The results indicate that DJOC was shown to be a promising adsorbent for the removal of Cr(VI) and Zn(II) ions from aqueous solution. PMID:23812789

  13. Indian craniometric variability and affinities.

    PubMed

    Raghavan, Pathmanathan; Bulbeck, David; Pathmanathan, Gayathiri; Rathee, Suresh Kanta

    2013-01-01

    Recently published craniometric and genetic studies indicate a predominantly indigenous ancestry of Indian populations. We address this issue with a fuller coverage of Indian craniometrics than any done before. We analyse metrical variability within Indian series, Indians' sexual dimorphism, differences between northern and southern Indians, index-based differences of Indian males from other series, and Indians' multivariate affinities. The relationship between a variable's magnitude and its variability is log-linear. This relationship is strengthened by excluding cranial fractions and series with a sample size less than 30. Male crania are typically larger than female crania, but there are also shape differences. Northern Indians differ from southern Indians in various features including narrower orbits and less pronounced medial protrusion of the orbits. Indians resemble Veddas in having small crania and similar cranial shape. Indians' wider geographic affinities lie with "Caucasoid" populations to the northwest, particularly affecting northern Indians. The latter finding is confirmed from shape-based Mahalanobis-D distances calculated for the best sampled male and female series. Demonstration of a distinctive South Asian craniometric profile and the intermediate status of northern Indians between southern Indians and populations northwest of India confirm the predominantly indigenous ancestry of northern and especially southern Indians. PMID:24455409

  14. Indian Craniometric Variability and Affinities

    PubMed Central

    Raghavan, Pathmanathan; Bulbeck, David; Pathmanathan, Gayathiri; Rathee, Suresh Kanta

    2013-01-01

    Recently published craniometric and genetic studies indicate a predominantly indigenous ancestry of Indian populations. We address this issue with a fuller coverage of Indian craniometrics than any done before. We analyse metrical variability within Indian series, Indians' sexual dimorphism, differences between northern and southern Indians, index-based differences of Indian males from other series, and Indians' multivariate affinities. The relationship between a variable's magnitude and its variability is log-linear. This relationship is strengthened by excluding cranial fractions and series with a sample size less than 30. Male crania are typically larger than female crania, but there are also shape differences. Northern Indians differ from southern Indians in various features including narrower orbits and less pronounced medial protrusion of the orbits. Indians resemble Veddas in having small crania and similar cranial shape. Indians' wider geographic affinities lie with “Caucasoid” populations to the northwest, particularly affecting northern Indians. The latter finding is confirmed from shape-based Mahalanobis-D distances calculated for the best sampled male and female series. Demonstration of a distinctive South Asian craniometric profile and the intermediate status of northern Indians between southern Indians and populations northwest of India confirm the predominantly indigenous ancestry of northern and especially southern Indians. PMID:24455409

  15. Fluorescent refrigeration

    DOEpatents

    Epstein, R.I.; Edwards, B.C.; Buchwald, M.I.; Gosnell, T.R.

    1995-09-05

    Fluorescent refrigeration is based on selective radiative pumping, using substantially monochromatic radiation, of quantum excitations which are then endothermically redistributed to higher energies. Ultimately, the populated energy levels radiatively deexcite emitting, on the average, more radiant energy than was initially absorbed. The material utilized to accomplish the cooling must have dimensions such that the exciting radiation is strongly absorbed, but the fluorescence may exit the material through a significantly smaller optical pathlength. Optical fibers and mirrored glasses and crystals provide this requirement. 6 figs.

  16. Fluorescence Microscopy

    PubMed Central

    Sanderson, Michael J.; Smith, Ian; Parker, Ian; Bootman, Martin D.

    2016-01-01

    Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. PMID:25275114

  17. Affine hypersurfaces with parallel difference tensor relative to affine α-connection

    NASA Astrophysics Data System (ADS)

    Li, Cece

    2014-12-01

    Li and Zhang (2014) studied affine hypersurfaces of R n + 1 with parallel difference tensor relative to the affine α-connection ∇ (α), and characterized the generalized Cayley hypersurfaces by K n - 1 ≠ 0 and ∇ (α) K = 0 for some nonzero constant α, where the affine α-connection ∇ (α) of information geometry was introduced on affine hypersurface. In this paper, by a slightly different method we continue to study affine hypersurfaces with ∇ (α) K = 0, if α = 0 we further assume that the Pick invariant vanishes and affine metric is of constant sectional curvature. It is proved that they are either hyperquadrics or improper affine hypersphere with flat indefinite affine metric, the latter can be locally given as a graph of a polynomial of at most degree n + 1 with constant Hessian determinant. In particular, if the affine metric is definite, Lorentzian, or its negative index is 2, we complete the classification of such hypersurfaces.

  18. A Zn(II)2Cys6 DNA binding protein regulates the sirodesmin PL biosynthetic gene cluster in Leptosphaeria maculans

    PubMed Central

    Fox, Ellen M.; Gardiner, Donald M.; Keller, Nancy P.; Howlett, Barbara J.

    2008-01-01

    A gene, sirZ, encoding a Zn(II)2Cys6 DNA binding protein is present in a cluster of genes responsible for the biosynthesis of the epipolythiodioxopiperazine (ETP) toxin, sirodesmin PL in the ascomycete plant pathogen, Leptosphaeria maculans. RNA-mediated silencing of sirZ gives rise to transformants that produce only residual amounts of sirodesmin PL and display a decrease in the transcription of several sirodesmin PL biosynthetic genes. This indicates that SirZ is a major regulator of this gene cluster. Proteins similar to SirZ are encoded in the gliotoxin biosynthetic gene cluster of Aspergillus fumigatus (gliZ) and in an ETP-like cluster in Penicillium lilacinoechinulatum (PlgliZ). Despite its high level of sequence similarity to gliZ, PlgliZ is unable to complement the gliotoxin-deficiency of a mutant of gliZ in A. fumigatus. Putative binding sites for these regulatory proteins in the promoters of genes in these clusters were predicted using bioinformatic analysis. These sites are similar to those commonly bound by other proteins with Zn(II)2Cys6 DNA binding domains. PMID:18023597

  19. Specific light exposure of galactosylated Zn(II) phthalocyanines for selective PDT effects on breast cancer cells

    NASA Astrophysics Data System (ADS)

    Mantareva, V. N.; Kril, A.; Angelov, I.; Avramov, L.

    2013-03-01

    Photodynamic therapy (PDT) is a clinically approved non-invasive and curative procedure for different oncological and non-oncological applications. PDT is still under development due to several limitations which lead to partially successful photodynamic response. The crucial steps in PDT procedure are binding of the photosensitizer to outer cell membrane, its penetration and subcellular localization which envisage the target sites of reactive oxygen species generated during irradiation. Since the surrounding normal cells are also exposed to the photosensitizer and the ambient daylight can be harmful for healthy tissues after therapeutic light application, the challenging task in PDT research is to optimize the procedure in a way to reach tumor cell selectivity. The present study outlines the influence of a light exposure pre-treatment (prior therapeutic light) with specific wavelengths (365 nm and 635 nm) on the uptake, the localization and further re-localization of galactose-substituted Zn(II) phthalocyanines into MDA-MB-231 breast cancer cells. The in vitro photodynamic effect towards tumor cells was studied in comparison to the normal cell line Balb/c 3T3 (clone 31) after pre-irradiation with UV light (365 nm) and red LED (635 nm). The results suggest that the galactose functional groups of Zn(II) phthalocyanine and the harmless UV light at 365 nm favor the selective PDT response.

  20. Nuclear-translocated endostatin downregulates hypoxia inducible factor-1α activation through interfering with Zn(II) homeostasis.

    PubMed

    Guo, Lifang; Chen, Yang; He, Ting; Qi, Feifei; Liu, Guanghua; Fu, Yan; Rao, Chunming; Wang, Junzhi; Luo, Yongzhang

    2015-05-01

    Hypoxia‑inducible factor‑1α (HIF‑1α) is key in tumor progression and aggressiveness as it regulates a series of genes involved in angiogenesis and anaerobic metabolism. Previous studies have shown that the transcriptional levels of HIF‑1α may be downregulated by endostatin. However, the molecular mechanism by which endostatin represses HIF‑1α expression remains unknown. The current study investigated the mechanism by which nuclear‑translocated endostatin suppresses HIF‑1α activation by disrupting Zn(II) homeostasis. Endostatin was observed to downregulate HIF‑1α expression at mRNA and protein levels. Blockage of endostatin nuclear translocation by RNA interference of importin α1/β1 or ectopic expression of NLS‑deficient mutant nucleolin in human umbilical vein endothelial cells co‑transfected with small interfering (si)‑nucleolin siRNA compromises endostatin‑reduced HIF‑1α expression. Nuclear‑translocated apo‑endostatin, but not holo‑endostatin, significantly disrupts the interaction between CBP/p300 and HIF‑1α by disturbing Zn(II) homeostasis, which leads to the transcriptional inactivation of HIF‑1α. The results reveal mechanistic insights into the method by which nuclear‑translocated endostatin downregulates HIF‑1α activation and provides a novel way to investigate the function of endostatin in endothelial cells. PMID:25607980

  1. The maximal affinity of ligands

    PubMed Central

    Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A.

    1999-01-01

    We explore the question of what are the best ligands for macromolecular targets. A survey of experimental data on a large number of the strongest-binding ligands indicates that the free energy of binding increases with the number of nonhydrogen atoms with an initial slope of ≈−1.5 kcal/mol (1 cal = 4.18 J) per atom. For ligands that contain more than 15 nonhydrogen atoms, the free energy of binding increases very little with relative molecular mass. This nonlinearity is largely ascribed to nonthermodynamic factors. An analysis of the dominant interactions suggests that van der Waals interactions and hydrophobic effects provide a reasonable basis for understanding binding affinities across the entire set of ligands. Interesting outliers that bind unusually strongly on a per atom basis include metal ions, covalently attached ligands, and a few well known complexes such as biotin–avidin. PMID:10468550

  2. Affinity chromatography based on a combinatorial strategy for rerythropoietin purification.

    PubMed

    Martínez-Ceron, María C; Marani, Mariela M; Taulés, Marta; Etcheverrigaray, Marina; Albericio, Fernando; Cascone, Osvaldo; Camperi, Silvia A

    2011-05-01

    Small peptides containing fewer than 10 amino acids are promising ligand candidates with which to build affinity chromatographic systems for industrial protein purification. The application of combinatorial peptide synthesis strategies greatly facilitates the discovery of suitable ligands for any given protein of interest. Here we sought to identify peptide ligands with affinity for recombinant human erythropoietin (rhEPO), which is used for the treatment of anemia. A combinatorial library containing the octapeptides X-X-X-Phe-X-X-Ala-Gly, where X = Ala, Asp, Glu, Phe, His, Leu, Asn, Pro, Ser, or Thr, was synthesized on HMBA-ChemMatrix resin by the divide-couple-recombine method. For the library screening, rhEPO was coupled to either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After cleavage, peptides were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fifty-seven beads showed a positive reaction. Peptides showing more consensuses were synthesized, and their affinity to rhEPO was assessed using a plasma resonance biosensor. Dissociation constant values in the range of 1-18 μM were obtained. The best two peptides were immobilized on Sepharose, and the resultant chromatographic matrixes showed affinity for rhEPO with dissociation constant values between 1.8 and 2.7 μM. Chinese hamster ovary (CHO) cell culture supernatant was spiked with rhEPO, and the artificial mixture was loaded on Peptide-Sepharose columns. The rhEPO was recovered in the elution fraction with a yield of 90% and a purity of 95% and 97% for P1-Sepharose and P2-Sepharose, respectively. PMID:21495625

  3. Protein Complex Purification by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Affinity capture has become a powerful technique for consistently purifying endogenous protein complexes, facilitating biochemical and biophysical assays on otherwise inaccessible biological assemblies, and enabling broader interactomic exploration. For this procedure, cells are broken and their contents separated and extracted into a solvent, permitting access to target macromolecular complexes thus released in solution. The complexes are specifically enriched from the extract onto a solid medium coupled with an affinity reagent-usually an antibody-that recognizes the target either directly or through an appended affinity tag, allowing subsequent characterization of the complex. Here, we discuss approaches and considerations for purifying endogenous yeast protein complexes by affinity capture. PMID:27371601

  4. A Novel Vertex Affinity for Community Detection

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  5. Structural determinants of sigma receptor affinity

    SciTech Connect

    Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

    1987-12-01

    The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

  6. Compact noncontraction semigroups of affine operators

    NASA Astrophysics Data System (ADS)

    Voynov, A. S.; Protasov, V. Yu

    2015-07-01

    We analyze compact multiplicative semigroups of affine operators acting in a finite-dimensional space. The main result states that every such semigroup is either contracting, that is, contains elements of arbitrarily small operator norm, or all its operators share a common invariant affine subspace on which this semigroup is contracting. The proof uses functional difference equations with contraction of the argument. We look at applications to self-affine partitions of convex sets, the investigation of finite affine semigroups and the proof of a criterion of primitivity for nonnegative matrix families. Bibliography: 32 titles.

  7. Tuning the structures of three coordination polymers incorporating ZnII and 2,2‧-dichloro-4,4‧-azodibenzoic acid via selective auxiliary ligands

    NASA Astrophysics Data System (ADS)

    Zeng, Xiao-Ping; Ming, Mei

    2015-11-01

    By tuning the auxiliary ligands in the assembling reaction, three ZnII coordination polymers of [Zn(Cl-adc) (phen) (H2O)](DMF) (1), [Zn(Cl-adc) (DMA)](DMA) (2), and [Zn(Cl-adc) (dip)](DMF)0.5 (3) (Cl-H2adc = 2,2‧-dichloro-4,4‧-azodibenzoic acid, phen = 1,10-phenanthroline, dip = 1,3-di(imidazole)propane) have been successfully synthesized and characterized by single-crystal X-ray diffraction study, elemental analysis, IR spectra, TGA analyses, solid-state fluorescent property, and powder X-ray diffraction (PXRD). Single crystal X-ray diffraction reveals that 1 and 2 displays a 1D polymeric chain and 2D sql layered net with the presence of chelated phen and terminal DMA ligands, respectively. By incorporating dip linker, 3 exhibits a 2D + 2D → 3D entangled network, with each 2D net portraying wavelike sql layered structure. Their structural divergences should be properly attributed to fact that, the structural topologies can be well regulated by using three auxiliary ligands incorparating different coordination function.

  8. Thiosemicarbazone Cu(II) and Zn(II) complexes as potential anticancer agents: syntheses, crystal structure, DNA cleavage, cytotoxicity and apoptosis induction activity.

    PubMed

    Shao, Jia; Ma, Zhong-Ying; Li, Ang; Liu, Ya-Hong; Xie, Cheng-Zhi; Qiang, Zhao-Yan; Xu, Jing-Yuan

    2014-07-01

    Four novel thiosemicarbazone metal complexes, [Cu(Am4M)(OAc)]·H2O (1), [Zn(HAm4M)Cl2] (2), [Zn2(Am4M)2Br2] (3) and [Zn2(Am4M)2(OAc)2]·2MeOH (4) [HAm4M=(Z)-2-(amino(pyridin-2-yl)methylene)-N-methylhydrazinecarbothioamide], have been synthesized and characterized by X-ray crystallography, elemental analysis, ESI-MS and IR. X-ray analysis revealed that complexes 1 and 2 are mononuclear, which possess residual coordination sites for Cu(II) ion in 1 and good leaving groups (Cl(-)) for Zn(II) ion in 2. Both 3 and 4 displayed dinuclear units, in which the metal atoms are doubly bridged by S atoms of two Am4M(-) ligands in 3 and by two acetate ions in bi- and mono-dentate forms, respectively, in 4. Their antiproliferative activities on human epithelial cervical cancer cell line (HeLa), human liver hepatocellular carcinoma cell line (HepG-2) and human gastric cancer cell line (SGC-7901) were screened. Inspiringly, IC50 value (11.2±0.9 μM) of complex 1 against HepG-2 cells was nearly 0.5 fold of that against human hepatic cell lines LO2, showing a lower toxicity to human liver cells. Additionally, it displayed a stronger inhibition on the viability of HepG-2 cells than cisplatin (IC50=25±3.1 μM), suggesting complex 1 might be a potential high efficient antitumor agent. Furthermore, fluorescence microscopic observation and flow cytometric analysis revealed that complex 1 could significantly suppress HepG-2 cell viability and induce apoptosis. Several indexes, such as DNA cleavage, reactive oxygen species (ROS) generation, comet assay and cell cycle analysis indicated that the antitumor mechanism of complex 1 on HepG-2 cells might be via ROS-triggered apoptosis pathway. PMID:24690556

  9. Structure of classical affine and classical affine fractional W-algebras

    SciTech Connect

    Suh, Uhi Rinn

    2015-01-15

    We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W-algebra via the complex. This definition clarifies that classical affine W-algebras can be considered as quasi-classical limits of quantum affine W-algebras. We also give a definition of a classical affine fractional W-algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W-algebra has two compatible λ-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W-algebra is associated to a minimal nilpotent, we describe explicit forms of free generators and compute λ-brackets between them. Provided some assumptions on a classical affine fractional W-algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel’d and Sokolov reduction.

  10. Cu(II) and Zn(II) ions alter the dynamics and distribution of Mn(II) in cultured chick glial cells

    SciTech Connect

    Wedler, F.C.; Ley, B.W. )

    1990-12-01

    Previous studies revealed that Mn(II) is accumulated in cultured glial cells to concentrations far above those present in whole brain or in culture medium. The data indicated that Mn(II) moves across the plasma membrane into the cytoplasm by facilitated diffusion or counter-ion transport with Ca(II), then into mitochondria by active transport. The fact that 1-10 microM Mn(II) ions activate brain glutamine synthetase makes important the regulation of Mn(II) transport in the CNS. Since Cu(II) and Zn(II) caused significant changes in the accumulation of Mn(II) by glia, the mechanisms by which these ions alter the uptake and efflux of Mn(II) ions has been investigated systematically under chemically defined conditions. The kinetics of (54MN)-Mn(II) uptake and efflux were determined and compared under four different sets of conditions: no adducts, Cu(II) or Zn(II) added externally, and with cells preloaded with Cu(II) or Zn(II) in the presence and absence of external added metal ions. Zn(II) ions inhibit the initial velocity of Mn(II) uptake, increase total Mn(II) accumulated, but do not alter the rate or extent Mn(II) efflux. Cu(II) ions increase both the initial velocity and the net Mn(II) accumulated by glia, with little effect on rate or extent of Mn(II) efflux. These results predict that increases in Cu(II) or Zn(II) levels may also increase the steady-state levels of Mn(II) in the cytoplasmic fraction of glial cells, which may in turn alter the activity of Mn(II)-sensitive enzymes in this cell compartment.

  11. Scaling analysis of affinity propagation.

    PubMed

    Furtlehner, Cyril; Sebag, Michèle; Zhang, Xiangliang

    2010-06-01

    We analyze and exploit some scaling properties of the affinity propagation (AP) clustering algorithm proposed by Frey and Dueck [Science 315, 972 (2007)]. Following a divide and conquer strategy we setup an exact renormalization-based approach to address the question of clustering consistency, in particular, how many cluster are present in a given data set. We first observe that the divide and conquer strategy, used on a large data set hierarchically reduces the complexity O(N2) to O(N((h+2)/(h+1))) , for a data set of size N and a depth h of the hierarchical strategy. For a data set embedded in a d -dimensional space, we show that this is obtained without notably damaging the precision except in dimension d=2 . In fact, for d larger than 2 the relative loss in precision scales such as N((2-d)/(h+1)d). Finally, under some conditions we observe that there is a value s* of the penalty coefficient, a free parameter used to fix the number of clusters, which separates a fragmentation phase (for ss*) of the underlying hidden cluster structure. At this precise point holds a self-similarity property which can be exploited by the hierarchical strategy to actually locate its position, as a result of an exact decimation procedure. From this observation, a strategy based on AP can be defined to find out how many clusters are present in a given data set. PMID:20866473

  12. Zn(II) and Cu(II) adsorption and retention onto iron oxyhydroxide nanoparticles: effects of particle aggregation and salinity

    PubMed Central

    2014-01-01

    Background Iron oxyhydroxides are commonly found in natural aqueous systems as nanoscale particles, where they can act as effective sorbents for dissolved metals due to their natural surface reactivity, small size and high surface area. These properties make nanoscale iron oxyhydroxides a relevant option for the remediation of water supplies contaminated with dissolved metals. However, natural geochemical processes, such as changes in ionic strength, pH, and temperature, can cause these particles to aggregate, thus affecting their sorption capabilities and remediation potential. Other environmental parameters such as increasing salinity may also impact metal retention, e.g. when particles are transported from freshwater to seawater. Results After using synthetic iron oxyhydroxide nanoparticles and nanoparticle aggregates in batch Zn(II) adsorption experiments, the addition of increasing concentrations of chloride (from 0.1 M to 0.6 M) appears to initially reduce Zn(II) retention, likely due to the desorption of outer-sphere zinc surface complexes and subsequent formation of aqueous Zn-Cl complexes, before then promoting Zn(II) retention, possibly through the formation of ternary surface complexes (supported by EXAFS spectroscopy) which stabilize zinc on the surface of the nanoparticles/aggregates. In batch Cu(II) adsorption experiments, Cu(II) retention reaches a maximum at 0.4 M chloride. Copper-chloride surface complexes are not indicated by EXAFS spectroscopy, but there is an increase in the formation of stable aqueous copper-chloride complexes as chloride concentration rises (with CuCl+ becoming dominant in solution at ~0.5 M chloride) that would potentially inhibit further sorption or encourage desorption. Instead, the presence of bidentate edge-sharing and monodentate corner-sharing complexes is supported by EXAFS spectroscopy. Increasing chloride concentration has more of an impact on zinc retention than the mechanism of nanoparticle aggregation, whereas

  13. Methods for Improving Aptamer Binding Affinity.

    PubMed

    Hasegawa, Hijiri; Savory, Nasa; Abe, Koichi; Ikebukuro, Kazunori

    2016-01-01

    Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of aptamers. In particular, sequence optimization using combined in silico sequence recombinations and in vitro functional evaluations is effective for the improvement of binding affinities, however, the binding affinities of aptamers are limited by the low hydrophobicity of nucleic acids. Accordingly, introduction of hydrophobic moieties into aptamers expands the diversity of interactions between aptamers and targets. Moreover, construction of multivalent aptamers by connecting aptamers that recognize distinct epitopes is an attractive approach to substantial increases in binding affinity. In addition, binding affinities can be tuned by optimizing the scaffolds of multivalent constructs. In this review, we summarize the various techniques for improving the binding affinities of aptamers. PMID:27043498

  14. Affine root systems and dual numbers

    NASA Astrophysics Data System (ADS)

    Kostyakov, I. V.; Gromov, N. A.; Kuratov, V. V.

    The root systems in Carroll spaces with degenerate metric are defined. It is shown that their Cartan matrices and reflection groups are affine. Due to the geometric consideration the root system structure of affine algebras is determined by a sufficiently simple algorithm.

  15. Loop realizations of quantum affine algebras

    SciTech Connect

    Cautis, Sabin; Licata, Anthony

    2012-12-15

    We give a simplified description of quantum affine algebras in their loop presentation. This description is related to Drinfeld's new realization via halves of vertex operators. We also define an idempotent version of the quantum affine algebra which is suitable for categorification.

  16. Improving image segmentation by learning region affinities

    SciTech Connect

    Prasad, Lakshman; Yang, Xingwei; Latecki, Longin J

    2010-11-03

    We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

  17. The future of fluorescence sensor arrays.

    PubMed

    Demchenko, Alexander P

    2005-09-01

    The rapid progress in sensor and biosensor array technologies needs a general strategy in the design of fluorescence reporters. Such reporters should provide a high density of sensor elements, allow analysis of targets of different affinities, and be internally calibrated, reproducible and have a rapid readout. Several criteria are introduced here for the comparative evaluation of fluorescence-sensing techniques. It is shown that only the two-band wavelength ratiometric sensing with a single reporter dye exhibiting rapid reversible excited-state reaction can satisfy all these criteria and is a prospective candidate for further development. PMID:15967523

  18. Pb(II)-promoted amide cleavage: mechanistic comparison to a Zn(II) analogue.

    PubMed

    Elton, Eric S; Zhang, Tingting; Prabhakar, Rajeev; Arif, Atta M; Berreau, Lisa M

    2013-10-01

    Two new Pb(II) complexes of the amide-appended nitrogen/sulfur epppa (N-((2-ethylthio)ethyl)-N-((6-pivaloylamido-2-pyridyl)methyl)-N-((2-pyridyl)methyl)amine) chelate ligand, [(epppa)Pb(NO3)2] (4-NO3) and [(epppa)Pb(ClO4)2] (4-ClO4), were prepared and characterized. In the solid state, 4-NO3 exhibits κ(5)-epppa chelate ligand coordination as well as the coordination of two bidentate nitrate ions. In acetonitrile, 4-NO3 is a 1:1 electrolyte with a coordinated NO3(-), whereas 4-ClO4 is a 1:2 electrolyte. Treatment of 4-ClO4 with 1 equiv Me4NOH·5H2O in CH3CN:CH3OH (3:5) results in amide methanolysis in a reaction that is akin to that previously reported for the Zn(II) analogue [(epppa)Zn](ClO4)2 (3-ClO4). (1)H NMR kinetic studies of the amide methanolysis reactions of 4-ClO4 and 3-ClO4 as a function of temperature revealed free energies of activation of 21.3 and 24.5 kcal/mol, respectively. The amide methanolysis reactions of 4-ClO4 and 3-ClO4 differ in terms of the effect of the concentration of methanol (saturation kinetics for 4-ClO4; second-order behavior for 3-ClO4), the observation of a small solvent kinetic isotope effect (SKIE) only for the reaction of the Zn(II)-containing 3-ClO4, and the properties of an initial intermediate isolated from each reaction upon treatment with Me4NOH·5H2O. These experimental results, combined with computational studies of the amide methanolysis reaction pathways of 4-ClO4 and 3-ClO4, indicate that the Zn(II)-containing 3-ClO4 initially undergoes amide deprotonation upon treatment with Me4NOH·5H2O. Subsequent amide protonation from coordinated methanol yields a structure containing a coordinated neutral amide and methoxide anion from which amide cleavage can then proceed. The rate-determining step in this pathway is either amide protonation or protonation of the leaving group. The Pb(II)-containing 4-ClO4 instead directly forms a neutral amide-containing, epppa-ligated Pb(II)-OH/Pb(II)-OCH3 equilibrium mixture upon treatment

  19. Improving Binding Affinity and Selectivity of Computationally Designed Ligand-Binding Proteins Using Experiments.

    PubMed

    Tinberg, Christine E; Khare, Sagar D

    2016-01-01

    The ability to de novo design proteins that can bind small molecules has wide implications for synthetic biology and medicine. Combining computational protein design with the high-throughput screening of mutagenic libraries of computationally designed proteins is emerging as a general approach for creating binding proteins with programmable binding modes, affinities, and selectivities. The computational step enables the creation of a binding site in a protein that otherwise does not (measurably) bind the intended ligand, and targeted mutagenic screening allows for validation and refinement of the computational model as well as provides orders-of-magnitude increases in the binding affinity. Deep sequencing of mutagenic libraries can provide insights into the mutagenic binding landscape and enable further affinity improvements. Moreover, in such a combined computational-experimental approach where the binding mode is preprogrammed and iteratively refined, selectivity can be achieved (and modulated) by the placement of specified amino acid side chain groups around the ligand in defined orientations. Here, we describe the experimental aspects of a combined computational-experimental approach for designing-using the software suite Rosetta-proteins that bind a small molecule of choice and engineering, using fluorescence-activated cell sorting and high-throughput yeast surface display, high affinity and ligand selectivity. We illustrated the utility of this approach by performing the design of a selective digoxigenin (DIG)-binding protein that, after affinity maturation, binds DIG with picomolar affinity and high selectivity over structurally related steroids. PMID:27094290

  20. Petasis-Ugi ligands: New affinity tools for the enrichment of phosphorylated peptides.

    PubMed

    Batalha, Íris L; Roque, Ana C A

    2016-09-15

    Affinity chromatography is a widespread technique for the enrichment and isolation of biologics, which relies on the selective and reversible interaction between affinity ligands and target molecules. Small synthetic affinity ligands are valuable alternatives due to their robustness, low cost and fast ligand development. This work reports, for the first time, the use of a sequential Petasis-Ugi multicomponent reaction to generate rationally designed solid-phase combinatorial libraries of small synthetic ligands, which can be screened for the selection of new affinity adsorbents towards biological targets. As a proof of concept, the Petasis-Ugi reaction was here employed in the discovery of affinity ligands suitable for phosphopeptide enrichment. A combinatorial library of 84 ligands was designed, synthesized on a chromatographic solid support and screened in situ for the specific binding of phosphopeptides binding human BRCA1C-terminal domains. The success of the reaction on the chromatographic matrix was confirmed by both inductively coupled plasma atomic emission spectroscopy and fluorescence microscopy. Three lead ligands were identified due to their superior performance in terms of binding capacity and selectivity towards the phosphorylated moiety on peptides, which showed the feasibility of the Petasis-Ugi reaction for affinity ligand development. PMID:27469904

  1. Fluorescent Human EP3 Receptor Antagonists.

    PubMed

    Tomasch, Miriam; Schwed, J Stephan; Kuczka, Karina; Meyer Dos Santos, Sascha; Harder, Sebastian; Nüsing, Rolf M; Paulke, Alexander; Stark, Holger

    2012-09-13

    Exchange of the lipophilc part of ortho-substituted cinnamic acid lead structures with different small molecule fluorophoric moieties via a dimethylene spacer resulted in hEP3R ligands with affinities in the nanomolar concentration range. Synthesized compounds emit fluorescence in the blue, green, and red range of light and have been tested concerning their potential as a pharmacological tool. hEP3Rs were visualized by confocal laser scanning microscopy on HT-29 cells, on murine kidney tissues, and on human brain tissues and functionally were characterized as antagonists on human platelets. Inhibition of PGE2 and collagen-induced platelet aggregation was measured after preincubation with novel hEP3R ligands. The pyryllium-labeled ligand 8 has been shown as one of the most promising structures, displaying a useful fluorescence and highly affine hEP3R antagonists. PMID:24900547

  2. An Optical Biosensor from Green Fluorescent Escherichia coli for the Evaluation of Single and Combined Heavy Metal Toxicities

    PubMed Central

    Futra, Dedi; Heng, Lee Yook; Ahmad, Asmat; Surif, Salmijah; Ling, Tan Ling

    2015-01-01

    A fluorescence-based fiber optic toxicity biosensor based on genetically modified Escherichia coli (E. coli) with green fluorescent protein (GFP) was developed for the evaluation of the toxicity of several hazardous heavy metal ions. The toxic metals include Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(III). The optimum fluorescence excitation and emission wavelengths of the optical biosensor were 400 ± 2 nm and 485 ± 2 nm, respectively. Based on the toxicity observed under optimal conditions, the detection limits of Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(III) that can be detected using the toxicity biosensor were at 0.04, 0.32, 0.46, 2.80, 100, 250, 400, 720 and 2600 μg/L, respectively. The repeatability and reproducibility of the proposed biosensor were 3.5%–4.8% RSD (relative standard deviation) and 3.6%–5.1% RSD (n = 8), respectively. The biosensor response was stable for at least five weeks, and demonstrated higher sensitivity towards metal toxicity evaluation when compared to a conventional Microtox assay. PMID:26029952

  3. Tuning structural topologies of two new luminescent Zn(II) coordination polymers via varying organic carboxylate ligands

    NASA Astrophysics Data System (ADS)

    Zhang, Yan; Liu, Qi-Feng; Xing, Guang'en; Zhang, Zhong-Qiang

    2015-04-01

    Presented here are two new luminescent Zn(II) coordination polymers, [Zn3(pdc)3(Hmtz)]n (1) and [Zn3(ntd)(mtz)4(DMA)2]n (2) (H2pdc = terephthalic acid, H2ntd = 2,6-naphthalenedicarboxylic acid, Hmtz = 5-methyl-1H-tetrazole). Single crystal X-ray diffraction analysis reveals that compound 1 features a 8-connected hex topological framework with the schläfli symbol of {36.418.53.6}, while compound 2 features a (3,4)-connected tfi topological framework with the schläfli symbol of {62.84}{62.8}2. The thermal stabilities and luminescent properties of 1-2 were also investigated.

  4. Recovery and concentration of metal ions. 4: Uphill transport of Zn(II) in a multimembrane hybrid system

    SciTech Connect

    Wodzki, R.; Sionkowski, G.; Pozniak, G.

    1999-02-01

    A study has been made on the uphill transport of zinc cations across a multimembrane hybrid system (MHS) composed of two ion-exchange membranes (IEM) separated by a bulk liquid membrane (BLM). The fluxes of the Zn(II)/H countertransport were investigated as dependent on the composition and structure of ion-exchange polymer membranes (i), the solvent of a liquid membrane (II), the feed and strip membrane area ratio (iii), and the pH of the feed solution (iv). The IEMs of various ionogenic groups (sulfonic acid, carboxylic acid, quaternized amine) and of various structure (clustered, gelatinous, porous) were examined in the MHS containing the BLM with di(2-ethylhexyl)phosphoric acid as a carrier of Zn(II) cations. It has been found that the Zn(II) fluxes are dependent on the properties of both the BLM and polymer membranes, i.e., on the BLM solvent viscosity (i), the nature and concentration of the IEM ion-exchange sites (ii), and the IEM thickness (iii). The best results were obtained when using hexane as the BLM solvent and the Nafion-117 membrane (perfluorinated polymer, sulfonic acid groups) as the cation-exchange membrane (CEM). The influence of the area ratio (feed-to-strip interface) has been checked for A{sub f}/A{sub g} equal to 3:1, 1:1, and 1:3. It was found that the asymmetry of the system leads mainly to some changes in the accumulation of transported species in a liquid membrane phase.

  5. Thermodynamic Study of CO2 Sorption by Polymorphic Microporous MOFs with Open Zn(II) Coordination Sites.

    PubMed

    Ahrenholtz, Spencer R; Landaverde-Alvarado, Carlos; Whiting, Macauley; Lin, Shaoyang; Slebodnick, Carla; Marand, Eva; Morris, Amanda J

    2015-05-01

    Two Zn-based metal organic frameworks have been prepared solvothermally, and their selectivity for CO2 adsorption was investigated. In both frameworks, the inorganic structural building unit is composed of Zn(II) bridged by the 2-carboxylate or 5-carboxylate pendants of 2,5-pyridine dicarboxylate (pydc) to form a 1D zigzag chain. The zigzag chains are linked by the bridging 2,5-carboxylates across the Zn ions to form 3D networks with formulas of Zn4(pydc)4(DMF)2·3DMF (1) and Zn2(pydc)2(DEF) (2). The framework (1) contains coordinated DMF as well as DMF solvates (DMF = N,N-dimethylformamide), while (2) contains coordinated DEF (DEF = N,N-diethylformamide). (1) displays a reversible type-I sorption isotherm for CO2 and N2 with BET surface areas of 196 and 319 m(2)/g, respectively. At low pressures, CO2 and N2 isotherms for (2) were not able to reach saturation, indicative of pore sizes too small for the gas molecules to penetrate. A solvent exchange to give (2)-MeOH allowed for increased CO2 and N2 adsorption onto the MOF surface with BET surface areas of 41 and 39 m(2)/g, respectively. The binding of CO2 into the framework of (1) was found to be exothermic with a zero coverage heat of adsorption, Qst(0), of −27.7 kJ/mol. The Qst(0) of (2) and (2)-MeOH were found to be −3 and −41 kJ/mol, respectively. The CO2/N2 selectivity for (1), calculated from the estimated KH at 296 K, was found to be 42. At pressures relevant to postcombustion capture, the selectivity was 14. The thermodynamic data are consistent with a mechanism of adsorption that involves CO2 binding to the unsaturated Zn(II) metal centers present in the crystal structures. PMID:25898142

  6. Kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum ATPase.

    PubMed

    Orlowski, S; Champeil, P

    1991-01-15

    We investigated the kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum Ca2(+)-ATPase by combining fast filtration with stopped-flow fluorescence measurements. At pH 6 and 20 degrees C, in the absence of potassium and in the presence of 20 mM MgCl2, isotopic exchange of bound calcium exhibited biphasic kinetics, with two phases of equal amplitude, regardless of the initial extent of binding site saturation. The rapidly exchangeable site, whose occupancy by calcium controlled the rate constant of the slow phase, had an apparent affinity for calcium of about 3-6 microM. A similar high affinity was also deduced from measurements of the calcium dependence of the rate constant for ATPase fluorescence changes. This affinity was higher than the overall affinity for calcium deduced from the equilibrium binding measurements (dissociation constant of 15-20 microM); this was consistent with the occurrence of cooperativity (Hill coefficient of 1.6-1.8). The drop in intrinsic fluorescence observed upon chelation of calcium was always slightly faster than the dissociation of calcium itself, although the rates for both this drop in fluorescence and calcium dissociation varied slightly from one preparation to the other. This fluorescence drop was therefore mainly due to dissociation of the bound ions, not to slow transconformation of the ATPase. Dissociation of the two bound calcium ions in a medium containing EGTA exhibited monophasic kinetics in the presence of a calcium ionophore, with a rate constant about half that of the fast phase of isotopic exchange. This particular pattern was observed over a wide range of experimental conditions, including the presence of KCl, dimethyl sulfoxide, 4-nonylphenol, or a nucleotide analogue, at pH 6 or 7, and at various temperatures. The kinetics of calcium dissociation under the above various conditions were not correlated with the ATPase affinity for calcium deduced from equilibrium

  7. Affinity Proteomics in the mountains: Alpbach 2015.

    PubMed

    Taussig, Michael J

    2016-09-25

    The 2015 Alpbach Workshop on Affinity Proteomics, organised by the EU AFFINOMICS consortium, was the 7th workshop in this series. As in previous years, the focus of the event was the current state of affinity methods for proteome analysis, including complementarity with mass spectrometry, progress in recombinant binder production methods, alternatives to classical antibodies as affinity reagents, analysis of proteome targets, industry focus on biomarkers, and diagnostic and clinical applications. The combination of excellent science with Austrian mountain scenery and winter sports engender an atmosphere that makes this series of workshops exceptional. The articles in this Special Issue represent a cross-section of the presentations at the 2015 meeting. PMID:27118167

  8. Optimized Affinity Capture of Yeast Protein Complexes.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Here, we describe an affinity isolation protocol. It uses cryomilled yeast cell powder for producing cell extracts and antibody-conjugated paramagnetic beads for affinity capture. Guidelines for determining the optimal extraction solvent composition are provided. Captured proteins are eluted in a denaturing solvent (sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer) for gel-based proteomic analyses. Although the procedures can be modified to use other sources of cell extract and other forms of affinity media, to date we have consistently obtained the best results with the method presented. PMID:27371596

  9. Aptamers in Affinity Separations: Stationary Separation

    NASA Astrophysics Data System (ADS)

    Ravelet, Corinne; Peyrin, Eric

    The use of DNA or RNA aptamers as tools in analytical chemistry is a very promising field of research because of their capabilities to bind specifically the target molecules with an affinity similar to that of antibodies. Notably, they appear to be of great interest as target-specific ligands for the separation and capture of various analytes in affinity chromatography and related affinity-based methods such as magnetic bead technology. In this chapter, the recent developments of these aptamer-based separation/capture approaches are addressed.

  10. Affinity purification of heme-tagged proteins.

    PubMed

    Asher, Wesley B; Bren, Kara L

    2014-01-01

    Protein affinity purification techniques are widely used for isolating pure target proteins for biochemical and structural characterization. Herein, we describe the protocol for affinity-based purification of proteins expressed in Escherichia coli that uses the coordination of a peptide tag covalently modified with heme c, known as a heme-tag, to an L-histidine immobilized Sepharose resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. In addition, we describe methods for specifically detecting heme-tagged proteins in SDS-PAGE gels using a heme-staining procedure and for quantifying the proteins using a pyridine hemochrome assay. PMID:24943311

  11. Method and apparatus for detection of fluorescently labeled materials

    DOEpatents

    Stern, David; Fiekowsky, Peter

    1997-01-01

    Fluorescently marked targets bind to a substrate 230 synthesized with polymer sequences at known locations. The targets are detected by exposing selected regions of the substrate 230 to light from a light source 100 and detecting the photons from the light fluoresced therefrom, and repeating the steps of exposure and detection until the substrate 230 is completely examined. The resulting data can be used to determine binding affinity of the targets to specific polymer sequences.

  12. Method and apparatus for detection of fluorescently labeled materials

    DOEpatents

    Stern, David; Fiekowsky, Peter

    2004-05-25

    Fluorescently marked targets bind to a substrate 230 synthesized with polymer sequences at known locations. The targets are detected by exposing selected regions of the substrate 230 to light from a light source 100 and detecting the photons from the light fluoresced therefrom, and repeating the steps of exposure and detection until the substrate 230 is completely examined. The resulting data can be used to determine binding affinity of the targets to specific polymer sequences.

  13. Zinspy Sensors with Enhanced Dynamic Range for Imaging Neuronal Cell Zinc Uptake and Mobilization

    PubMed Central

    Nolan, Elizabeth M.; Ryu, Jubin W.; Jaworski, Jacek; Feazell, Rodney P.; Sheng, Morgan; Lippard, Stephen J.

    2006-01-01

    Thiophene moieties were incorporated into previously described Zinspy (ZS) fluorescent Zn(II) sensor motifs (Nolan, E. M.; Lippard, S. J. Inorg. Chem. 2004, 43, 8310–8317) to provide enhanced fluorescence properties, low-micromolar dissociation constants for Zn(II), and improved Zn(II) selectivity. Halogenation of the xanthenone and benzoate moieties of the fluorescein platform systematically modulates the excitation and emission profiles, pH-dependent fluorescence, Zn(II) affinity, and Zn(II) complexation rates, offering a general strategy for tuning multiple properties of xanthenone-based metal ion sensors. Extensive biological studies in cultured cells and primary neuronal cultures demonstrate 2-{6-hydroxy-3-oxo-4,5-bis[(pyridin-2-ylmethylthiophen-2-ylmethylamino)methyl]-3H-xanthen-9-yl}benzoic acid (ZS5) to be a versatile imaging tool for detecting Zn(II) in vivo. ZS5 localizes to the mitochondria of HeLa cells and allows visualization of glutamate-mediated Zn(II) uptake in dendrites and Zn(II) release resulting from nitrosative stress in neurons. PMID:17132019

  14. PRINCIPLES OF AFFINITY-BASED BIOSENSORS

    EPA Science Inventory

    Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

  15. Minimal information to determine affine shape equivalence.

    PubMed

    Wagemans, J; Van Gool, L; Lamote, C; Foster, D H

    2000-04-01

    Participants judged the affine equivalence of 2 simultaneously presented 4-point patterns. Performance level (d') varied between 1.5 and 2.7, depending on the information available for solving the correspondence problem (insufficient in Experiment 1a, superfluous in Experiment 1b, and minimal in Experiments 1c, 2a, 2b) and on the exposure time (unlimited in Experiments 1 and 2a and 500 ms in Experiment 2b), but it did not vary much with the complexity of the affine transformation (rotation and slant in Experiment 1 and same plus tilt in Experiment 2). Performance in Experiment 3 was lower with 3-point patterns than with 4-point patterns, whereas blocking the trials according to the affine transformation parameters had little effect. Determining affine shape equivalence with minimal-information displays is based on a fast assessment of qualitatively or quasi-invariant properties such as convexity/ concavity, parallelism, and collinearity. PMID:10811156

  16. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-01-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. PMID:25829303

  17. Visualizing antibody affinity maturation in germinal centers.

    PubMed

    Tas, Jeroen M J; Mesin, Luka; Pasqual, Giulia; Targ, Sasha; Jacobsen, Johanne T; Mano, Yasuko M; Chen, Casie S; Weill, Jean-Claude; Reynaud, Claude-Agnès; Browne, Edward P; Meyer-Hermann, Michael; Victora, Gabriel D

    2016-03-01

    Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with nonimmunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  18. The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144

    SciTech Connect

    Briers, Yves; Schmelcher, Mathias; Loessner, Martin J.; Hendrix, Jelle; Engelborghs, Yves; Volckaert, Guido; Lavigne, Rob

    2009-05-29

    The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD{sub KZ}), originating from Pseudomonas aeruginosa bacteriophage {phi}KZ, has been examined using a fusion protein of PBD{sub KZ} and green fluorescent protein (PBD{sub KZ}-GFP). A fluorescence recovery after photobleaching analysis of bound PBD{sub KZ}-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 x 10{sup 7} M{sup -1} for the PBD{sub KZ}-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD{sub KZ}-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.

  19. Use of quantitative affinity chromatography for characterizing high-affinity interactions: binding of heparin to antithrombin III.

    PubMed

    Hogg, P J; Jackson, C M; Winzor, D J

    1991-02-01

    The versatility of quantitative affinity chromatography (QAC) for evaluating the binding of macromolecular ligands to macromolecular acceptors has been increased substantially as a result of the derivation of the equations which describe the partitioning of acceptor between matrix-bound and soluble forms in terms of total, rather than free, ligand concentrations. In addition to simplifying the performance of the binding experiments, this development makes possible the application of the technique to systems characterized by affinities higher than those previously amenable to investigation by QAC. Addition of an on-line data acquisition system to monitor the concentration of partitioning solute in the liquid phase as a function of time has permitted the adoption of an empirical approach for determining the liquid-phase concentration of acceptor in the system at partition equilibrium, a development which decreases significantly the time required to obtain a complete binding curve by QAC. The application of these new QAC developments is illustrated by the determination of binding constants for the interactions of high-affinity heparin (Mr 20,300) with antithrombin III at three temperatures. Association constants of 8.0 +/- 2.2 x 10(7), 3.4 +/- 0.3 x 10(7), and 1.0 +/- 0.2 x 10(7) M-1 were observed at 15, 25, and 35 degrees C, respectively. The standard enthalpy change of -4.2 +/- 0.6 kcal/mol that is calculated from these data is in good agreement with a reported value obtained from fluorescence quenching measurements. PMID:2035830

  20. Infrared and Raman spectra of ethylene trithiocarbonate complexes of some Zn(II), Cd(II) and Hg(II) halides

    NASA Astrophysics Data System (ADS)

    Contreras, J. Guillermo; Gnecco, Juan A.

    Coordination compounds of ethylene trithiocarbonate (ETTC) with some Zn(II), Cd(II) and Hg(II) halides have been prepared, characterized and their infrared and Raman spectra recorded. The i.r. spectra in the range 4000-400 cm -1 suggest that the organic ligand is bonded to the metal ions through its exocyclic sulphur atom, whereas the far-i.r. and Raman spectra show that the complexes of the type HgX 2(ETTC) (X = Cl, Br or I) possess a trans dimeric halogen-bridged structure. The Cd(II) and Zn(II) species are of the type MX 2(ETTC) 2 and they possess a pseudotetrahedral structure of C2υ symmetry.

  1. Synthesis, Characterization and Biological Evaluation of Co(II), Cu(II), Ni(II) and Zn(II) Complexes With Cephradine

    PubMed Central

    Jaffery, Maimoon F.

    2000-01-01

    Some Co(II), Cu(II), Ni(II) and Zn(II) complexes of antibacterial drug cephradine have been prepared and characterized by their physical, spectral and analytical data. Cephradine acts as bidentate and the complexes have compositions, [M(L)2X2] where [M = Co(II), Ni(II) and Zn(II), L = cephradine and X = Cl2] showing octahedral geometry, and [M(L)2] where [M = Cu(II), L = cephradine] showing square planar geometry. In order to evaluate the effect of metal ions upon chelation, eephradine and its complexes have been screened for their antibacterial activity against bacterial strains, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. PMID:18475955

  2. NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ...

  3. Diarylethene based fluorescent switchable probes for the detection of amyloid-β pathology in Alzheimer's disease.

    PubMed

    Lv, Guanglei; Cui, Baiping; Lan, Haichuang; Wen, Ying; Sun, Anyang; Yi, Tao

    2015-01-01

    Two fluorescent switchable diarylethene derivatives which exhibit high affinity for amyloid-β aggregates with the increase of fluorescence intensity were reported. Moreover, the probes show excellent photochromic and anti-photobleaching properties both in vitro and in vivo. PMID:25384304

  4. NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ALDH2 catalyzes oxidation of toxic aldehydes to their corresponding carboxylic acids. Magnesium ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements have monitored the blue shift of the NADH fluorescence spectrum to elucidate the extent of...

  5. Affinity engineering of maltoporin: variants with enhanced affinity for particular ligands.

    PubMed

    Clune, A; Lee, K S; Ferenci, T

    1984-05-31

    Affinity-chromatographic selection on immobilized starch was used to selectively enhance the affinity of the maltodextrin-specific pore protein ( maltoporin , LamB protein, or lambda receptor protein) in the outer membrane of E. coli. Selection strategies were established for rare bacteria in large populations producing maltoporin variants with enhanced affinities for both starch and maltose, for starch but not maltose and for maltose but not starch. Three classes of lamB mutants with up to eight-fold increase in affinity for particular ligands were isolated. These mutants provide a unique range of modifications in the specificity of a transport protein. PMID:6375667

  6. Genome-wide analysis of the Zn(II)2Cys6 zinc cluster-encoding gene family in Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins with a Zn(II)2Cys6 domain, Cys-X2-Cys-X6-Cys-X5-12-Cys-X2-Cys-X6-9-Cys (hereafter, referred to as the C6 domain), form a subclass of zinc finger proteins found exclusively in fungi and yeast. Genome sequence databases of Saccharomyces cerevisiae and Candida albicans have provided an overvie...

  7. Sensitive electrochemical sensor using a graphene-polyaniline nanocomposite for simultaneous detection of Zn(II), Cd(II), and Pb(II).

    PubMed

    Ruecha, Nipapan; Rodthongkum, Nadnudda; Cate, David M; Volckens, John; Chailapakul, Orawon; Henry, Charles S

    2015-05-18

    This work describes the development of an electrochemical sensor for simultaneous detection of Zn(II), Cd(II), and Pb(II) using a graphene-polyaniline (G/PANI) nanocomposite electrode prepared by reverse-phase polymerization in the presence of polyvinylpyrrolidone (PVP). Two substrate materials (plastic film and filter paper) and two nanocomposite deposition methods (drop-casting and electrospraying) were investigated. Square-wave anodic stripping voltammetry currents were higher for plastic vs. paper substrates. Performance of the G/PANI nanocomposites was characterized by scanning electron microscopy (SEM) and cyclic voltammetry. The G/PANI-modified electrode exhibited high electrochemical conductivity, producing a three-fold increase in anodic peak current (vs. the unmodified electrode). The G/PANI-modified electrode also showed evidence of increased surface area under SEM. Square-wave anodic stripping voltammetry was used to measure Zn(II), Cd(II), and Pb(II) in the presence of Bi(III). A linear working range of 1-300 μg L(-1) was established between anodic current and metal ion concentration with detection limits (S/N=3) of 1.0 μg L(-1) for Zn(II), and 0.1 μg L(-1) for both Cd(II) and Pb(II). The G/PANI-modified electrode allowed selective determination of the target metals in the presence of common metal interferences including Mn(II), Cu(II), Fe(III), Fe(II), Co(III), and Ni(II). Repeat assays on the same device demonstrated good reproducibility (%RSD<11) over 10 serial runs. Finally, this system was utilized for determining Zn(II), Cd(II), and Pb(II) in human serum using the standard addition method. PMID:25910444

  8. Crystal structure of the plant dual-affinity nitrate transporter NRT1.1

    NASA Astrophysics Data System (ADS)

    Sun, Ji; Bankston, John R.; Payandeh, Jian; Hinds, Thomas R.; Zagotta, William N.; Zheng, Ning

    2014-03-01

    Nitrate is a primary nutrient for plant growth, but its levels in soil can fluctuate by several orders of magnitude. Previous studies have identified Arabidopsis NRT1.1 as a dual-affinity nitrate transporter that can take up nitrate over a wide range of concentrations. The mode of action of NRT1.1 is controlled by phosphorylation of a key residue, Thr 101 however, how this post-translational modification switches the transporter between two affinity states remains unclear. Here we report the crystal structure of unphosphorylated NRT1.1, which reveals an unexpected homodimer in the inward-facing conformation. In this low-affinity state, the Thr 101 phosphorylation site is embedded in a pocket immediately adjacent to the dimer interface, linking the phosphorylation status of the transporter to its oligomeric state. Using a cell-based fluorescence resonance energy transfer assay, we show that functional NRT1.1 dimerizes in the cell membrane and that the phosphomimetic mutation of Thr 101 converts the protein into a monophasic high-affinity transporter by structurally decoupling the dimer. Together with analyses of the substrate transport tunnel, our results establish a phosphorylation-controlled dimerization switch that allows NRT1.1 to uptake nitrate with two distinct affinity modes.

  9. Dicyanovinylnaphthalenes for neuroimaging of amyloids and relationships of electronic structures and geometries to binding affinities

    PubMed Central

    Petrič, Andrej; Johnson, Scott A.; Pham, Hung V.; Li, Ying; Čeh, Simon; Golobič, Amalija; Agdeppa, Eric D.; Timbol, Gerald; Liu, Jie; Keum, Gyochang; Satyamurthy, Nagichettiar; Kepe, Vladimir; Houk, Kendall N.; Barrio, Jorge R.

    2012-01-01

    The positron-emission tomography (PET) probe 2-(1-[6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene) (FDDNP) is used for the noninvasive brain imaging of amyloid-β (Aβ) and other amyloid aggregates present in Alzheimer’s disease and other neurodegenerative diseases. A series of FDDNP analogs has been synthesized and characterized using spectroscopic and computational methods. The binding affinities of these molecules have been measured experimentally and explained through the use of a computational model. The analogs were created by systematically modifying the donor and the acceptor sides of FDDNP to learn the structural requirements for optimal binding to Aβ aggregates. FDDNP and its analogs are neutral, environmentally sensitive, fluorescent molecules with high dipole moments, as evidenced by their spectroscopic properties and dipole moment calculations. The preferred solution-state conformation of these compounds is directly related to the binding affinities. The extreme cases were a nonplanar analog t-butyl-FDDNP, which shows low binding affinity for Aβ aggregates (520 nM Ki) in vitro and a nearly planar tricyclic analog cDDNP, which displayed the highest binding affinity (10 pM Ki). Using a previously published X-ray crystallographic model of 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP) bound to an amyloidogenic Aβ peptide model, we show that the binding affinity is inversely related to the distortion energy necessary to avoid steric clashes along the internal surface of the binding channel. PMID:23012452

  10. Fluorescence of dental porcelain.

    PubMed

    Monsénégo, G; Burdairon, G; Clerjaud, B

    1993-01-01

    This study of the fluorescence of natural enamel and of dental ceramics shows the fluorescence of ceramics not containing rare earths decreases when the color saturation increases; the fluorescence of samples of the same shade guide are not homogenous; some guides show a strong green fluorescence; and two shade guides of the same origin can present completely different fluorescence. The cementing medium can affect the fluorescence of a ceramic prosthesis. PMID:8455155

  11. Biosorption of Cu(II), Zn(II), Ni(II) and Pb(II) ions by cross-linked metal-imprinted chitosans with epichlorohydrin.

    PubMed

    Chen, Chia-Yun; Yang, Cheng-Yu; Chen, Arh-Hwang

    2011-03-01

    Cross-linked metal-imprinted chitosan microparticles were prepared from chitosan, using four metals (Cu(II), Zn(II), Ni(II), and Pb(II)) as templates, and epichlorohydrin as the cross-linker. The microparticles were characterized by Fourier transform infrared spectroscopy, solid state (13)C nuclear magnetic resonance spectroscopy, and energy-dispersive X-ray spectroscopy. They were used for comparative biosorption of Cu(II), Zn(II), Ni(II) and Pb(II) ions in an aqueous solution. The results showed that the sorption capacities of Cu(II), Zn(II), Ni(II), and Pb(II) on the templated microparticles increased from 25 to 74%, 13 to 46%, 41 to 57%, and 12 to 43%, respectively, as compared to the microparticles without metal ion templates. The dynamic study showed that the sorption process followed the second-order kinetic equation. Three sorption models, Langmuir, Freundlich, and Dubinin-Radushkevich, were applied to the equilibrium isotherm data. The result showed that the Langmuir isotherm equation best fitted for monolayer sorption processes. Furthermore, the microparticles can be regenerated and reused for the metal removal. PMID:21044814

  12. Ni(II) and Zn(II) complexes of 2-((thiophen-2-ylmethylene)amino)benzamide: Synthesis, spectroscopic characterization, thermal, DFT and anticancer activities

    NASA Astrophysics Data System (ADS)

    Tyagi, Prateek; Chandra, Sulekh; Saraswat, B. S.

    2015-01-01

    The paper presents the synthesis of Ni(II) and Zn(II) complexes of general composition M(L)X2 and M(L)2X2 (M = Ni(II), Zn(II), X = Cl-1, OAc-1) with Schiff base obtained through the condensation of 2-aminobenzamide with thiophene-2-carbaldehyde. The characterization of newly formed complexes was done by 1H NMR, UV-VIS, TGA, IR, mass spectrophotometry and molar conductivity studies. The thermal studies suggested that the complexes are more stable as compared to ligand. In DFT studies the geometries of Schiff's base and metal complexes were fully optimized with respect to the energy using the 6-31+g(d,p) basis set. On the basis of the spectral studies a distorted octahedral geometry has been assigned for Ni(II) complexes and tetrahedral geometry for Zn(II) complexes. The effect of these complexes on proliferation of human breast cancer cell line (MCF-7) and human hepatocellular liver carcinoma cell line (HepG2) were studied and compared with those of free ligand.

  13. Preparation of cross-linked magnetic chitosan-phenylthiourea resin for adsorption of Hg(II), Cd(II) and Zn(II) ions from aqueous solutions.

    PubMed

    Monier, M; Abdel-Latif, D A

    2012-03-30

    In this study, cross-linked magnetic chitosan-phenylthiourea (CSTU) resin were prepared and characterized by means of FTIR, (1)H NMR, SEM high-angle X-ray diffraction (XRD), magnetic properties and thermogravimetric analysis (TGA). The prepared resin were used to investigate the adsorption properties of Hg(II), Cd(II) and Zn(II) metal ions in an aqueous solution. The extent of adsorption was investigated as a function of pH and the metal ion removal reached maximum at pH 5.0. Also, the kinetic and thermodynamic parameters of the adsorption process were estimated. These data indicated that the adsorption process is exothermic and followed the pseudo-second-order kinetics. Equilibrium studies showed that the data of Hg(II), Cd(II) and Zn(II) adsorption followed the Langmuir model. The maximum adsorption capacities for Hg(II), Cd(II) and Zn(II) were estimated to be 135 ± 3, 120 ± 1 and 52 ± 1 mg/g, which demonstrated the high adsorption efficiency of CSTU toward the studied metal ions. PMID:22277339

  14. Simultaneous determination of Cd(II), Cu(II), Pb(II), and Zn(II) in citrus essential oils by derivative potentiometric stripping analysis.

    PubMed

    La Pera, Lara; Saitta, Marcello; Di Bella, Giuseppa; Dugo, Giacomo

    2003-02-26

    Citrus essential oils are widely used in the food, cosmetics, and pharmaceutical industries, so the determination of heavy metals content is of great importance to guarantee their quality. The present work deals with the quantification of Cd(II), Cu(II), Pb(II), and Zn(II) in different varieties of citrus essential oils, using derivative potentiometric stripping analysis. Two different metals extraction procedures, involving concentrated hydrochloric acid treatment and acid-alcoholic dissolution, are tested on lemon, mandarin, sweet orange, and bergamot essential oils, and they give very similar results. Cd(II), Cu(II), Pb(II), and Zn(II) recovery tests spanned from 95 to 100.50%, providing evidence that metals quantification remained unaffected by the cleanup steps of the two procedures. The repeatability of the hydrochloric acid extraction method, applied on different varieties of essential oils, is >95.00% for Cd(II), Cu(II), Pb(II), and Zn(II), whereas the repeatability of the acid-alcoholic dissolution method is >93.00% for Cu and Cd only in lemon oil. Detection limits obtained for the four analytes, using both procedures, ranged from 0.10 to 0.98 ng g(-)(1) in lemon, mandarin, sweet orange, and bergamot essential oils. PMID:12590445

  15. Synthesis, characterization, DFT and biological studies of isatinpicolinohydrazone and its Zn(II), Cd(II) and Hg(II) complexes

    NASA Astrophysics Data System (ADS)

    El-Gammal, O. A.; Rakha, T. H.; Metwally, H. M.; Abu El-Reash, G. M.

    2014-06-01

    Isatinpicolinohydrazone (H2IPH) and its Zn(II), Cd(II) and Hg(II) complexes have been synthesized and investigated using physicochemical techniques viz. IR, 1H NMR, 13C NMR, UV-Vis spectrometric methods and magnetic moment measurements. The investigation revealed that H2IPH acts as binegative tetradentate in Zn(II), neutral tridentate in Cd(II) and as neutral bidentate towards Hg(II) complex. Octahedral geometry is proposed for all complexes. The bond length, bond angle, chemical reactivity, energy components (kcal/mol), binding energy (kcal/mol) and dipole moment (Debyes) for all the title compounds were evaluated by DFT and also MEP for the ligand is shown. Theoretical infrared intensities of H2IPH and also the theoretical electronic spectra of the ligand and its complexes were calculated. The thermal behavior and the kinetic parameters of degradation were determined using Coats-Redfern and Horowitz-Metzger methods. The in vitro antibacterial studies of the complexes proved them as growth inhibiting agents. The DDPH antioxidant of the compounds have been screened. Antitumor activity, carried out in vitro on human mammary gland (breast) MCF7, have shown that Hg(II) complex exhibited potent activity followed by Zn(II), Cd(II) complexes and the ligand.

  16. Binding properties and structure-affinity relationships of food antioxidant butylated hydroxyanisole and its metabolites with lysozyme.

    PubMed

    Wu, Di; Yan, Jin; Tang, Peixiao; Li, Shanshan; Xu, Kailin; Li, Hui

    2015-12-01

    Considering the harmful impact of food antioxidants on human bodies, thoroughly exposing their potential effects at the molecular level is important. In this study, the binding interactions of butylated hydroxyanisole (BHA), a phenolic antioxidant, and its different major metabolites tert-butylhydroquinone (TBHQ) and tert-butylbenzoquinone (TBQ) with lysozyme were examined via fluorescence, three-dimensional fluorescence, circular dichroism (CD), and ligand-protein docking studies. The three compounds caused strong quenching of lysozyme fluorescence by a static quenching mechanism but with different quenching efficiencies and different effects on the α-helix content of the lysozyme. The order of binding affinity of lysozyme for all test compounds is as follows: BHA>TBQ>TBHQ. Thermodynamic parameters indicated that hydrogen bonding and van der Waals forces perform dominant functions in the binding between these compounds and lysozyme. Furthermore, structure-affinity relationships between the model compounds and lysozyme were established on the basis of computational analyses. PMID:26041206

  17. Four Zn(II)/Cd(II)-3-amino-1,2,4-triazolate frameworks constructed by in situ metal/ligand reactions: Structures and fluorescent properties

    SciTech Connect

    Chen Zilu; Li Xiaoling; Liang Fupei

    2008-08-15

    Four Cd(II) and Zn(II) complexes with the in situ-generated ligand of 3-amino-1,2,4-triazolate (AmTAZ{sup -}) were isolated from the solvothermal reactions of the corresponding Cd(II) or Zn(II) salts with 5-amino-1H-1,2,4-triazole-3-carboxylic acid (AmTAZAc). Their structures were determined by single-crystal X-ray diffraction analysis. [Zn(AmTAZ)(CH{sub 3}COO)] (1) presents a two-dimensional framework constructed from Zn(II) ions and {mu}{sub 3}-AmTAZ{sup -} ligands. A remarkable feature of [Zn{sub 4}(AmTAZ){sub 4}(SO{sub 4})(OH)(C{sub 2}O{sub 4}){sub 0.5}].2H{sub 2}O (2) is the construction of the building units of octagonal cylinders which interact with each other by sharing one face or overlapping, resulting in the formation of a three-dimensional framework with three kinds of 1D channels. [Cd(AmTAZ)Br] (3) crystallizes in a chiral space group P2{sub 1}2{sub 1}2{sub 1}, giving a homochiral three-dimensional framework with two types of helical channels (left- and right-handed). Different from the others, the 3-amino-1,2,4-triazole molecules in [Cd(AmTAZH)SO{sub 4}] (4) behave as neutral {mu}{sub 2}-2,4-bridges to connect the two-dimensional CdSO{sub 4} sheets into a three-dimensional framework. Of all, 2 and 3 display different fluorescent properties probably due to different metal ions, coordination environments and structural topologies. - Graphical abstract: The solvothermal reactions of Cd(II) and Zn(II) salts bearing different anions with 5-amino-1H-1,2,4-triazole-3-carboxylic acid (AmTAZAc) produced four Cd(II) and Zn(II) MOFs with the in situ-generated 3-amino-1,2,4-triazolate (AmTAZ{sup -}) ion as ligand, which display different structural topologies and fluorescent properties. Display Omitted.

  18. Classification of neocortical interneurons using affinity propagation

    PubMed Central

    Santana, Roberto; McGarry, Laura M.; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  19. Classification of neocortical interneurons using affinity propagation.

    PubMed

    Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  20. Affinity purification of aprotinin from bovine lung.

    PubMed

    Xin, Yu; Liu, Lanhua; Chen, Beizhan; Zhang, Ling; Tong, Yanjun

    2015-05-01

    An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration, and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25,476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15,907.1 ± 10.2 kallikrein inactivator unit/mg. PMID:25677462

  1. Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

    ERIC Educational Resources Information Center

    Parsons, Julie; Ridley, Kimberly

    2012-01-01

    Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same time,…

  2. Characterizing new fluorescent tools for studying 5-HT₃ receptor pharmacology.

    PubMed

    Jack, Thomas; Simonin, Jonathan; Ruepp, Marc-David; Thompson, Andrew J; Gertsch, Jürg; Lochner, Martin

    2015-03-01

    The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP; 1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron, tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with co-crystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo live imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology, including FC and FP ligand competition, to live imaging of 5-HT3 expressing tissues. PMID:25460187

  3. Tailoring Fluorescent Dyes To Optimize a Hybrid RGD-Tracer.

    PubMed

    Bunschoten, Anton; van Willigen, Danny M; Buckle, Tessa; van den Berg, Nynke S; Welling, Mick M; Spa, Silvia J; Wester, Hans-Jürgen; van Leeuwen, Fijs W B

    2016-05-18

    Quantitative assessment of affinity and kinetics is a critical component in the development of (receptor-targeted) radiotracers. For fluorescent tracers, such an assessment is currently not yet applied, while (small) changes in chemical composition of the fluorescent component might have substantial influence on the overall properties of a fluorescent tracer. Hybrid imaging labels that contain both a radiolabel and a fluorescent dye can be used to evaluate both the affinity (fluorescent label) and the in vivo distribution (radiolabel) of a targeted tracer. We present a hybrid label oriented and matrix-based scoring approach that enabled quantitative assessment of the influence of (overall) charge and lipophilicity of the fluorescent label on the (in vivo) characteristics of αvβ3-integrin targeted tracers. Systematic chemical alterations in the fluorescent dye were shown to result in a clear difference in the in vivo distribution of the different hybrid tracers. The applied evaluation technique resulted in an optimized targeted tracer for αvβ3-integrin, which combined the highest T/M ratio with the lowest uptake in other organs. Obviously this selection concept would also be applicable during the development of other (receptor-targeted) imaging tracers. PMID:27074375

  4. Indole-7-carbaldehyde thiosemicarbazone as a flexidentate ligand toward ZnII, CdII, PdII and PtII ions: cytotoxic and apoptosis-inducing properties of the PtII complex.

    PubMed

    Ibrahim, Abeer A; Khaledi, Hamid; Hassandarvish, Pouya; Mohd Ali, Hapipah; Karimian, Hamed

    2014-03-14

    A new thiosemicarbazone (LH2) derived from indole-7-carbaldehyde was synthesized and reacted with Zn(II), Cd(II), Pd(II) and Pt(II) salts. The reactions with zinc and cadmium salts in 2 : 1 (ligand-metal) molar ratio afforded complexes of the type MX2(LH2)2, (X = Cl, Br or OAc), in which the thiosemicarbazone acts as a neutral S-monodentate ligand. In the presence of potassium hydroxide, the reaction of LH2 with ZnBr2 resulted in deprotonation of the thiosemicarbazone at the hydrazine and indole nitrogens to form Zn(L)(CH3OH). The reaction of LH2 with K2PdCl4 in the presence of triethylamine, afforded Pd(L)(LH2) which contains two thiosemicarbazone ligands: one being dianionic N,N,S-tridentate while the other one is neutral S-monodentate. When PdCl2(PPh3)2 was used as the Pd(II) ion source, Pd(L)(PPh3) was obtained. In a similar manner, the analogous platinum complex, Pt(L)(PPh3), was synthesized. The thiosemicarbazone in the latter two complexes behaves in a dianionic N,N,S-tridentate fashion. The platinum complex was found to have significant cytotoxicity toward four cancer cells lines, namely MDA-MB-231, MCF-7, HT-29, and HCT-116 but not toward the normal liver WRL-68 cell line. The apoptosis-inducing properties of the Pt complex was explored through fluorescence microscopy visualization, DNA fragmentation analysis and propidium iodide flow cytometry. PMID:24442181

  5. A new Salen-type azo-azomethine ligand and its Ni(II), Cu(II) and Zn(II) complexes: Synthesis, spectral characterization, crystal structure and photoluminescence studies.

    PubMed

    Ozkan, Gozde; Kose, Muhammet; Zengin, Huseyin; McKee, Vickie; Kurtoglu, Mukerrem

    2015-11-01

    A novel Salen-type azo-azomethine ligand H2agen, 2,2'-{ethane-1,2-diylbis[nitrilomethylylidene]}bis{4-[ethylphenyldiazenyl]phenol}, formed by the 1:2M condensation of ethane-1,2-diamine with 5-[(4-ethylphenyl)diazenyl]-2-hydroxybenzaldehyde and its nickel(II), copper(II), and zinc(II) complexes were synthesized and characterized by the spectroscopic and analytical methods. The UV-vis spectra of the ligand were investigated in three organic solvents (DMSO, DMF and CHCl3). The ligand shows two absorption bands assigned to π-π(∗) and n-π(∗) transitions in the solvents used. Cu(II), and Ni(II) are tetra-coordinate binding to two phenolic oxygens and two imine nitrogens in approximate square planar geometry. Zn(II) also coordinates using the same sites like other metals but gave tetragonal configuration. Molecular structure of the Cu(II) complex [Cu(agen)] was determined by single crystal X-ray diffraction study. The X-ray data revealed that crystallographic imposed symmetry was absent for the complex molecule. In the structure, the Cu(II) ion is coordinated to two phenolate oxygen atoms and two imine nitrogen atoms of the azo-azomethine ligand with approximate square planar geometry. The ligand H2agen and its metal complexes exhibit strong blue emissions with irradiation. Fluorescence quantum yields and excited-state lifetimes for the ligand and its complexes were obtained. The H2agen ligand had a 35% quantum yield and a 3.27 ns excited-state lifetime. Complexation with metal ions caused reductions in intensities and quantum yields. PMID:26123514

  6. On Affine Fusion and the Phase Model

    NASA Astrophysics Data System (ADS)

    Walton, Mark A.

    2012-11-01

    A brief review is given of the integrable realization of affine fusion discovered recently by Korff and Stroppel. They showed that the affine fusion of the su(n) Wess-Zumino-Novikov-Witten (WZNW) conformal field theories appears in a simple integrable system known as the phase model. The Yang-Baxter equation leads to the construction of commuting operators as Schur polynomials, with noncommuting hopping operators as arguments. The algebraic Bethe ansatz diagonalizes them, revealing a connection to the modular S matrix and fusion of the su(n) WZNW model. The noncommutative Schur polynomials play roles similar to those of the primary field operators in the corresponding WZNW model. In particular, their 3-point functions are the su(n) fusion multiplicities. We show here how the new phase model realization of affine fusion makes obvious the existence of threshold levels, and how it accommodates higher-genus fusion.

  7. The dynamics of metric-affine gravity

    SciTech Connect

    Vitagliano, Vincenzo; Sotiriou, Thomas P.; Liberati, Stefano

    2011-05-15

    Highlights: > The role and the dynamics of the connection in metric-affine theories is explored. > The most general second order action does not lead to a dynamical connection. > Including higher order invariants excites new degrees of freedom in the connection. > f(R) actions are also discussed and shown to be a non- representative class. - Abstract: Metric-affine theories of gravity provide an interesting alternative to general relativity: in such an approach, the metric and the affine (not necessarily symmetric) connection are independent quantities. Furthermore, the action should include covariant derivatives of the matter fields, with the covariant derivative naturally defined using the independent connection. As a result, in metric-affine theories a direct coupling involving matter and connection is also present. The role and the dynamics of the connection in such theories is explored. We employ power counting in order to construct the action and search for the minimal requirements it should satisfy for the connection to be dynamical. We find that for the most general action containing lower order invariants of the curvature and the torsion the independent connection does not carry any dynamics. It actually reduces to the role of an auxiliary field and can be completely eliminated algebraically in favour of the metric and the matter field, introducing extra interactions with respect to general relativity. However, we also show that including higher order terms in the action radically changes this picture and excites new degrees of freedom in the connection, making it (or parts of it) dynamical. Constructing actions that constitute exceptions to this rule requires significant fine tuned and/or extra a priori constraints on the connection. We also consider f(R) actions as a particular example in order to show that they constitute a distinct class of metric-affine theories with special properties, and as such they cannot be used as representative toy theories to

  8. Displacement phenomena in lectin affinity chromatography.

    PubMed

    Cho, Wonryeon

    2015-10-01

    The work described here examines displacement phenomena that play a role in lectin affinity chromatography and their potential to impact reproducibility. This was achieved using Lycopersicon esculentum lectin (LEL), a lectin widely used in monitoring cancer. Four small identical LEL columns were coupled in series to form a single affinity chromatography system with the last in the series connected to an absorbance detector. The serial affinity column set (SACS) was then loaded with human plasma proteins. At the completion of loading, the column set was disassembled, the four columns were eluted individually, the captured proteins were trypsin digested, the peptides were deglycosylated with PNGase F, and the parent proteins were identified through mass spectral analyses. Significantly different sets of glycoproteins were selected by each column, some proteins appearing to be exclusively bound to the first column while others were bound further along in the series. Clearly, sample displacement chromatography (SDC) occurs. Glycoproteins were bound at different places in the column train, identifying the presence of glycoforms with different affinity on a single glycoprotein. It is not possible to see these phenomena in the single column mode of chromatography. Moreover, low abundance proteins were enriched, which facilitates detection. The great advantage of this method is that it differentiates between glycoproteins on the basis of their binding affinity. Displacement phenomena are concluded to be a significant component of the separation mechanism in heavily loaded lectin affinity chromatography columns. This further suggests that care must be exercised in sample loading of lectin columns to prevent analyte displacement with nonretained proteins. PMID:26348026

  9. Synthesis, characterization, crystal structure and cytotoxic properties of thiosemicarbazide Ni(II) and Zn(II) complexes

    NASA Astrophysics Data System (ADS)

    Mathan Kumar, S.; Rajesh, J.; Anitha, K.; Dhahagani, K.; Marappan, M.; Indra Gandhi, N.; Rajagopal, G.

    2015-05-01

    Synthesis of new complexes of Ni(II) (1) and Zn(II) (2) with [1-(2-hydroxy-3,5-diiodobenzylidene)-4-phenylthiosemicarbazide] have been reported. The composition of these two complexes 1 and 2 is discussed on the basis of IR, 1H NMR and UV spectral data along with their X-ray crystallographic data. The crystal structure of these two complexes has revealed that the free ligand (L) is deprotonated twice at the oxygen and sulfur atoms and they are coordinated with the complexes through phenoxide-O, azomethine-N and thiolate-S atoms. The single-crystal X-ray structures of complex (1) exhibits a square planar structure, while complex (2) reveals trigonal bipyramidal distorted square based pyramidal structure. Anticancer activity of ligand and the complexes 1-2 are evaluated in human adenocarcinoma (MCF-7) cells. The preliminary bioassay indicates that the free ligand and the complexes 1-2 exhibit inhibitory activity against the human adenocarcinoma cancer cell lines.

  10. Spectroscopy, NMR and DFT studies on molecular recognition of crown ether bridged chiral heterotrinuclear salen Zn(II) complex

    NASA Astrophysics Data System (ADS)

    Gao, Feng; Ruan, Wen-Juan; Chen, Jia-Mei; Zhang, Ying-Hui; Zhu, Zhi-Ang

    2005-12-01

    A barium-containing crown ether bridged chiral heterotrinuclear salen Zn(II) complex BaZn 2L(ClO 4) 2, where L is a folded dinuclear chiral ( R, R)-salen ligand, has been synthesized and characterized by elemental analysis, 1H NMR, UV-vis, IR, circular dichroism (CD) spectra, and mass spectra. As a folded dinuclear chiral host, its recognition with achiral guests (imidazole derivatives), rigid bidentate guest (1,4-diazobicyclo[2,2,2]octane, DABCO) and chiral guests (amino acid methyl esters) was investigated by means of UV-vis spectrophotometric titration, CD spectra. The association constants of D-amino acid methyl esters are found to be higher than those of their L-enantiomer. The sandwich-type binding of BaZn 2L(ClO 4) 2-DABCO supramolecular assembly was specially studied via 1H NMR titration and 1H ROESY. To understand the recognition on molecular level, density functional theory (DFT) calculations on B3LYP/LanL2DZ were performed on the minimal energy conformations of host, guests, and host-guest complexes. The minimal energy conformations were obtained by molecular mechanics (MM) optimization and molecular dynamics (MD) simulation. The results of single point energy, HOMO energy, and charges transfer were analyzed. The results of theoretical calculations are in good agreement with the experimental data.

  11. Synthesis, characterization, crystal structure and cytotoxic properties of thiosemicarbazide Ni(II) and Zn(II) complexes.

    PubMed

    Mathan Kumar, S; Rajesh, J; Anitha, K; Dhahagani, K; Marappan, M; Indra Gandhi, N; Rajagopal, G

    2015-05-01

    Synthesis of new complexes of Ni(II) (1) and Zn(II) (2) with [1-(2-hydroxy-3,5-diiodobenzylidene)-4-phenylthiosemicarbazide] have been reported. The composition of these two complexes 1 and 2 is discussed on the basis of IR, (1)H NMR and UV spectral data along with their X-ray crystallographic data. The crystal structure of these two complexes has revealed that the free ligand (L) is deprotonated twice at the oxygen and sulfur atoms and they are coordinated with the complexes through phenoxide-O, azomethine-N and thiolate-S atoms. The single-crystal X-ray structures of complex (1) exhibits a square planar structure, while complex (2) reveals trigonal bipyramidal distorted square based pyramidal structure. Anticancer activity of ligand and the complexes 1-2 are evaluated in human adenocarcinoma (MCF-7) cells. The preliminary bioassay indicates that the free ligand and the complexes 1-2 exhibit inhibitory activity against the human adenocarcinoma cancer cell lines. PMID:25706599

  12. Ethylenediamine-modified graphene oxide covalently functionalized with a tetracarboxylic Zn(ii) phthalocyanine hybrid for enhanced nonlinear optical properties.

    PubMed

    Li, Zongle; He, Chunying; Wang, Zhao; Gao, Yachen; Dong, Yongli; Zhao, Cheng; Chen, Zhimin; Wu, Yiqun; Song, Weina

    2016-07-01

    Tetracarboxylic Zn(ii) phthalocyanine-amino functionalized graphene oxide (ZnPcC4-NGO) hybrid materials have been prepared by a covalent functionalization method. The characterizations indicate that the amino-functionalization of GO has an important influence on the structure and photophysical properties of the ZnPcC4-NGO hybrid. The ZnPcC4-NGO hybrid exhibits enhanced photo-induced electron transfer or energy transfer (PET/ET), compared to the ZnPcC4 covalent functionalized GO (ZnPcC4-GO), owing to the presence of the extended sp(2) carbon configurations, along with the partial reduction of the NGO nanosheets and the introduction of electron-donating ethylenediamine. The nonlinear optical (NLO) properties of the hybrids were investigated using the Z-scan technique at 532 nm with 4 ns laser pulses. The results show that the efficient covalent functionalization and partial reduction of NGO cause the ZnPcC4-NGO hybrid to possess evidently larger NLO properties than the individual NGO, ZnPcC4 and the ZnPcC4-GO hybrid. The enhanced NLO performance can be attributed to the increased excited state absorption from the extended sp(2) carbon configurations of the NGO moiety, reverse saturable absorption arising from ZnPcC4 moiety, and the contribution of the efficient PET/ET process between the ZnPcC4 and NGO moieties in the hybrid. PMID:27296527

  13. Negative Electron Affinity Mechanism for Diamond Surfaces

    NASA Technical Reports Server (NTRS)

    Krainsky, I. L.; Asnin, V. M.

    1998-01-01

    The energy distribution of the secondary electrons for chemical vacuum deposited diamond films with Negative Electron Affinity (NEA) was investigated. It was found that while for completely hydrogenated diamond surfaces the negative electron affinity peak in the energy spectrum of the secondary electrons is present for any energy of the primary electrons, for partially hydrogenated diamond surfaces there is a critical energy above which the peak is present in the spectrum. This critical energy increases sharply when hydrogen coverage of the diamond surface diminishes. This effect was explained by the change of the NEA from the true type for the completely hydrogenated surface to the effective type for the partially hydrogenated surfaces.

  14. New unitary affine-Virasoro constructions

    SciTech Connect

    Halpern, M.B.; Kiritsis, E.; Obers, N.A.; Poratti, M. ); Yamron, J.P. )

    1990-06-20

    This paper reports on a quasi-systematic investigation of the Virasoro master equation. The space of all affine-Virasoro constructions is organized by K-conjugation into affine-Virasoro nests, and an estimate of the dimension of the space shows that most solutions await discovery. With consistent ansatze for the master equation, large classes of new unitary nests are constructed, including quadratic deformation nests with continuous conformal weights, and unitary irrational central charge nests, which may dominate unitary rational central charge on compact g.

  15. Adsorption affinity of anions on metal oxyhydroxides

    NASA Astrophysics Data System (ADS)

    Pechenyuk, S. I.; Semushina, Yu. P.; Kuz'mich, L. F.

    2013-03-01

    The dependences of anion (phosphate, carbonate, sulfate, chromate, oxalate, tartrate, and citrate) adsorption affinity anions from geometric characteristics, acid-base properties, and complex forming ability are generalized. It is shown that adsorption depends on the nature of both the anions and the ionic medium and adsorbent. It is established that anions are generally grouped into the following series of adsorption affinity reduction: PO{4/3-}, CO{3/2-} > C2O{4/2-}, C(OH)(CH2)2(COO){3/3-}, (CHOH)2(COO){2/2-} > CrO{4/2-} ≫ SO{4/2-}.

  16. Unusual Recognition and Separation of Hydrated Metal Sulfates [M2(μ-SO4)2(H2O)n, M = Zn(II), Cd(II), Co(II), Mn(II)] by a Ditopic Receptor.

    PubMed

    Ghosh, Tamal Kanti; Dutta, Ranjan; Ghosh, Pradyut

    2016-04-01

    A ditopic receptor L1, having metal binding bis(2-picolyl) donor and anion binding urea group, is synthesized and explored toward metal sulfate recognition via formation of dinuclear assembly, (L1)2M2(SO4)2. Mass spectrometric analysis, (1)H-DOSY NMR, and crystal structure analysis reveal the existence of a dinuclear assembly of MSO4 with two units of L1. (1)H NMR study reveals significant downfield chemical shift of -NH protons of urea moiety of L1 selectively with metal sulfates (e.g., ZnSO4, CdSO4) due to second-sphere interactions of sulfate with the urea moiety. Variable-temperature (1)H NMR studies suggest the presence of intramolecular hydrogen bonding interaction toward metal sulfate recognition in solution state, whereas intermolecular H-bonding interactions are observed in solid state. In contrast, anions in their tetrabutylammonium salts fail to interact with the urea -NH probably due to poor acidity of the tertiary butyl urea group of L1. Metal sulfate binding selectivity in solution is further supported by isothermal titration calorimetric studies of L1 with different Zn salts in dimethyl sulfoxide (DMSO), where a binding affinity is observed for ZnSO4 (Ka = 1.23 × 10(6)), which is 30- to 50-fold higher than other Zn salts having other counteranions in DMSO. Sulfate salts of Cd(II)/Co(II) also exhibit binding constants in the order of ∼1 × 10(6) as in the case of ZnSO4. Positive role of the urea unit in the selectivity is confirmed by studying a model ligand L2, which is devoid of anion recognition urea unit. Structural characterization of four MSO4 [M = Zn(II), Cd(II), Co(II), Mn(II)] complexes of L1, that is, complex 1, [(L1)2(Zn)2(μ-SO4)2]; complex 2, [(L1)2(H2O)2(Cd)2(μ-SO4)2]; complex 3, [(L1)2(H2O)2(Co)2(μ-SO4)2]; and complex 4, [(L1)2(H2O)2(Mn)2(μ-SO4)2], reveal the formation of sulfate-bridged eight-membered crownlike binuclear complexes, similar to one of the concentration-dependent dimeric forms of MSO4 as observed in solid state

  17. Molecular Structure-Affinity Relationship of Bufadienolides and Human Serum Albumin In Vitro and Molecular Docking Analysis

    PubMed Central

    Wang, Honglan; Zhang, Junfeng; Duan, Jinao; Ma, Hongyue; Wu, Qinan

    2015-01-01

    The development of bufadienolides as anti-tumor agents is limited due to poor pharmacokinetic properties regarding drug half-lives and toxicity in vivo. These serious factors might be improved by increasing the drug/albumin-binding ratio. This study therefore investigated the relationship between the structural properties of nine bufadienolides and their affinities for human serum albumin (HSA) by a fluorescence spectroscopy-based analysis and molecular docking. Fluorescence quenching data showed that the interaction of each bufadienolide with HSA formed a non-fluorescent complex, while thermodynamic parameters revealed negative ΔS and ΔH values, corresponding to changes in enthalpy and entropy, respectively. The structural differences between the various bufadienolides markedly influenced their binding affinity for HSA. With the exception of a C = O bond at the C12 position that decreased the binding affinity for HSA, other polar groups tended to increase the affinity, especially a hydroxyl (OH) group at assorted bufadienolide sites. The rank order of binding affinities for drugs with tri-hydroxyl groups was as follows: 11-OH > 5-OH > 16-OH; in addition, 16-acetoxy (OAc), 10-aldehyde and 14-epoxy constituents notably enhanced the binding affinity. Among these groups, 11-OH and 16-acetyl were especially important for a seamless interaction between the bufadienolides and HSA. Furthermore, molecular docking analysis revealed that either an 11-OH or a 16-OAc group spatially close to a five-membered lactone ring significantly facilitated the anchoring of these compounds within site I of the HSA pocket via hydrogen bonding (H-bonding) with Tyr150 or Lys199, respectively. In summary, bufadienolide structure strongly affects binding with HSA, and 11-OH or 16-OAc groups improve the drug association with key amino acid residues. This information is valuable for the prospective development of bufadienolides with improved pharmacological profiles as novel anti-tumor drugs

  18. Molecular structure-affinity relationship of bufadienolides and human serum albumin in vitro and molecular docking analysis.

    PubMed

    Zhou, Jing; Lu, Guodi; Wang, Honglan; Zhang, Junfeng; Duan, Jinao; Ma, Hongyue; Wu, Qinan

    2015-01-01

    The development of bufadienolides as anti-tumor agents is limited due to poor pharmacokinetic properties regarding drug half-lives and toxicity in vivo. These serious factors might be improved by increasing the drug/albumin-binding ratio. This study therefore investigated the relationship between the structural properties of nine bufadienolides and their affinities for human serum albumin (HSA) by a fluorescence spectroscopy-based analysis and molecular docking. Fluorescence quenching data showed that the interaction of each bufadienolide with HSA formed a non-fluorescent complex, while thermodynamic parameters revealed negative ΔS and ΔH values, corresponding to changes in enthalpy and entropy, respectively. The structural differences between the various bufadienolides markedly influenced their binding affinity for HSA. With the exception of a C = O bond at the C12 position that decreased the binding affinity for HSA, other polar groups tended to increase the affinity, especially a hydroxyl (OH) group at assorted bufadienolide sites. The rank order of binding affinities for drugs with tri-hydroxyl groups was as follows: 11-OH > 5-OH > 16-OH; in addition, 16-acetoxy (OAc), 10-aldehyde and 14-epoxy constituents notably enhanced the binding affinity. Among these groups, 11-OH and 16-acetyl were especially important for a seamless interaction between the bufadienolides and HSA. Furthermore, molecular docking analysis revealed that either an 11-OH or a 16-OAc group spatially close to a five-membered lactone ring significantly facilitated the anchoring of these compounds within site I of the HSA pocket via hydrogen bonding (H-bonding) with Tyr150 or Lys199, respectively. In summary, bufadienolide structure strongly affects binding with HSA, and 11-OH or 16-OAc groups improve the drug association with key amino acid residues. This information is valuable for the prospective development of bufadienolides with improved pharmacological profiles as novel anti-tumor drugs

  19. Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step.

    PubMed

    Huang, Renhua; Gorman, Kevin T; Vinci, Chris R; Dobrovetsky, Elena; Gräslund, Susanne; Kay, Brian K

    2015-01-01

    Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the "affinity maturation" step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold. PMID:26437402

  20. Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step

    PubMed Central

    Huang, Renhua; Gorman, Kevin T.; Vinci, Chris R.; Dobrovetsky, Elena; Gräslund, Susanne; Kay, Brian K.

    2015-01-01

    Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the “affinity maturation” step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold. PMID:26437402

  1. High-affinity lead binding proteins in rat kidney cytosol mediate cell-free nuclear translocation of lead

    SciTech Connect

    Mistry, P.; Lucier, G.W.; Fowler, B.A.

    1985-02-01

    The PbII binding characteristics of the previously reported PbII binding proteins of rat kidney cytosol were investigated further. Saturation and Scatchard analysis of /sup 203/Pb binding in whole cytosol and in 40% saturated ammonium sulfate precipitated fractions disclosed a class of relatively high-affinity sites with an apparent Kd of approximately 50 nM and binding capacities of approximately 41 and 9 pmol/mg of protein, respectively. Two /sup 203/Pb binding proteins with approximate molecular masses of 63K and 11.5K daltons and a high molecular weight component (greater than 200K) were isolated by Sepharose-6B column chromatography. The time course of association of /sup 203/Pb with cytosol and the 63K protein showed maximum binding at 18 hr which was stable up to 25 hr at 4 degrees C. The approximate half-time dissociation rate (T 1/2) of specifically bound /sup 203/Pb to the 63K protein was 100 min at 4 degrees C whereas the 11.5K protein showed little dissociation of specifically bound ligand at this temperature. Saturation analysis of the three isolated proteins disclosed low capacity, high-affinity sites with similar apparent Kd values to the cytosol assay. Sucrose density gradient analysis of kidney cytosol showed approximate sedimentation coefficients of 2S, 4.6S and 7S for the 11.5K, 63K and the high molecular weight proteins, respectively. Competitive binding studies with cytosol demonstrated displacement of /sup 203/Pb by PbII, CdII and ZnII ions but not CaII ions.

  2. Properties of impurity-bearing ferrihydrite II: Insights into the surface structure and composition of pure, Al- and Si-bearing ferrihydrite from Zn(II) sorption experiments and Zn K-edge X-ray absorption spectroscopy

    NASA Astrophysics Data System (ADS)

    Cismasu, A. Cristina; Levard, Clément; Michel, F. Marc; Brown, Gordon E.

    2013-10-01

    Naturally occurring ferrihydrite often contains impurities such as Al and Si, which can impact its chemical reactivity with respect to metal(loid) adsorption and (in)organic or microbially induced reductive dissolution. However, the surface composition of impure ferrihydrites is not well constrained, and this hinders our understanding of the factors controlling the surface reactivity of these nanophases. In this study, we conducted Zn(II) adsorption experiments combined with Zn K-edge X-ray absorption spectroscopy measurements on pure ferrihydrite (Fh) and Al- or Si-bearing ferrihydrites containing 10 and 20 mol% Al or Si (referred to as 10AlFh, 20AlFh and 10SiFh, 20SiFh) to evaluate Zn(II) uptake in relation to Zn(II) speciation at their surfaces. Overall, Zn(II) uptake at the surface of AlFh is similar to that of pure Fh, and based on Zn K-edge EXAFS data, Zn(II) speciation at the surface of Fh and AlFh also appears similar. Binuclear bidentate IVZn-VIFe complexes (at ∼3.46 Å (2C[1]) and ∼3.25 Å (2C[2])) were identified at low Zn(II) surface coverages from Zn K-edge EXAFS fits. With increasing Zn(II) surface coverage, the number of second-neighbor Fe ions decreased, which was interpreted as indicating the formation of IVZn polymers at the ferrihydrite surface, and a deviation from Langmuir uptake behavior. Zn(II) uptake at the surface of SiFh samples was more significant than at Fh and AlFh surfaces, and was attributed to the formation of outer-sphere complexes (on average 24% of sorbed Zn). Although similar Zn-Fe/Zn distances were obtained for the Zn-sorbed SiFh samples, the number of Fe second neighbors was lower in comparison with Fh. The decrease in second-neighbor Fe is most pronounced for sample 20SiFh, suggesting that the amount of reactive surface Fe sites diminishes with increasing Si content. Although our EXAFS results shown here do not provide evidence for the existence of Zn-Al or Zn-Si complexes, their presence is not excluded for Zn-sorbed Al

  3. High affinity of lead for fetal haemoglobin.

    PubMed Central

    Ong, C N; Lee, W R

    1980-01-01

    In-vitro experiments using 203Pb were performed to identify lead-binding components in human haemoglobin. Sephadex A-50 ion-exchange chromatography of haemolysate showed that different types of haemoglobin had different affinities for lead. For the haemolysate from adults, lead was present in both Hb A (alpha 2 beta 2) and Hb A2 (alpha 2 delta 2), whereas, in the haemolysate from new-born infants, the haemoglobin of fetal origin, Hb F (alpha 2 gamma 2) showed a much greater affinity for 203Pb than the adult haemoglobin Hb A (alpha 2 beta 2), obtained from maternal blood. Analysis of the 203 Pb-labelled haemoglobin suggested that about 82% of 203Pb was in the globin polypeptide. Further analysis with carboxylmethyl (CM) cellulose chromatography indicated that the gamma globin of fetal origin had a higher affinity for 203Pb than the beta globin, whereas alpha globin appeared to be unimportant in lead binding. The results of the different affinities for lead of different Hb types are discussed with regard to the effect of lead upon haemoglobin synthesis. PMID:6158989

  4. Vygotsky's and Buber's Pedagogical Perspectives: Some Affinities

    ERIC Educational Resources Information Center

    Bartholo, Roberto; Tunes, Elizabeth; Tacca, Maria Carmen Villela Rosa

    2010-01-01

    The purpose of this paper is to examine the dialogical and creative character of pedagogic work by analyzing the affinities between Martin Buber's "I-Thou relation" and Lev Semenovich Vygotsky's "Zone of Proximal Development". Backed up by empirical studies on the teacher-student relation, we understand that education can only result in students'…

  5. Fan Affinity Laws from a Collision Model

    ERIC Educational Resources Information Center

    Bhattacharjee, Shayak

    2012-01-01

    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour…

  6. Fundamentals of fluorescence and fluorescence microscopy.

    PubMed

    Wolf, David E

    2013-01-01

    This chapter discusses the fundamental physics of fluorescence. The application of fluorescence to microscopy represents an important transition in the development of microscopy, particularly as it applies to biology. It enables quantitating the amounts of specific molecules within a cell, determining whether molecules are complexing on a molecular level, measuring changes in ionic concentrations within cells and organelles, and measuring molecular dynamics. This chapter also discusses the issues important to quantitative measurement of fluorescence and focuses on four of quantitative measurements of fluorescence--boxcar-gated detection, streak cameras, photon correlation, and phase modulation. Although quantitative measurement presents many pitfalls to the beginner, it also presents significant opportunities to one skilled in the art. This chapter also examines how fluorescence is measured in the steady state and time domain and how fluorescence is applied in the modern epifluorescence microscope. PMID:23931503

  7. Binding affinities of CRBPI and CRBPII for 9-cis-retinoids

    PubMed Central

    Kane, Maureen A.; Bright, Frank V.; Napoli, Joseph L.

    2014-01-01

    Background Cellular retinol binding-protein I (CRBPI) and cellular retinol binding-protein II (CRBPII) serve as intracellular retinoid chaperones that bind retinol and retinal with high affinity and facilitate substrate delivery to select enzymes that catalyze retinoic acid (RA) and retinyl ester biosynthesis. Recently, 9-cis-RA has been identified in vivo in the pancreas, where it contributes to regulating glucose-stimulated insulin secretion. In vitro, 9-cis-RA activates RXR (retinoid×receptors), which serve as therapeutic targets for treating cancer and metabolic diseases. Binding affinities and structure–function relationships have been well characterized for CRBPI and CRBPII with all-trans-retinoids, but not for 9-cis-retinoids. This study extended current knowledge by establishing binding affinities for CRBPI and CRBPII with 9-cis-retinoids. Methods We have determined apparent dissociation constants, Kd′, through monitoring binding of 9-cis-retinol, 9-cis-retinal, and 9-cis-RA with CRBPI and CRBPII by fluorescence spectroscopy, and analyzing the data with non-linear regression. We compared these data to the data we obtained for all-trans- and 13-cis-retinoids under identical conditions. Results CRBPI and CRBPII, respectively, bind 9-cis-retinol ( Kd′, 11 nM and 68 nM) and 9-cis-retinal ( Kd′, 8 nM and 5 nM) with high affinity. No significant 9-cis-RA binding was observed with CRBPI or CRBPII. Conclusions CRBPI and CRBPII bind 9-cis-retinol and 9-cis-retinal with high affinities, albeit with affinities somewhat lower than for all-trans-retinol and all-trans-retinal. General significance These data provide further insight into structure–binding relationships of cellular retinol binding-proteins and are consistent with a model of 9-cis-RA biosynthesis that involves chaperoned delivery of 9-cis-retinoids to enzymes that recognize retinoid binding-proteins. PMID:21382444

  8. The Affinity of Cholesterol for Different Phospholipids Affects Lateral Segregation in Bilayers.

    PubMed

    Engberg, Oskar; Hautala, Victor; Yasuda, Tomokazu; Dehio, Henrike; Murata, Michio; Slotte, J Peter; Nyholm, Thomas K M

    2016-08-01

    Saturated and unsaturated phospholipids (PLs) can segregate into lateral domains. The preference of cholesterol for saturated acyl chains over monounsaturated, and especially polyunsaturated ones, may also affect lateral segregation. Here we have studied how cholesterol influenced the lateral segregation of saturated and unsaturated PLs, for which cholesterol had a varying degree of affinity. The fluorescence lifetime of trans-parinaric acid reported the formation of ordered domains (gel or liquid-ordered (lo)) in bilayers composed of different unsaturated phosphatidylcholines, and dipalmitoyl-phosphatidylcholine or n-palmitoyl-sphingomyelin, in the presence or absence of cholesterol. We observed that cholesterol facilitated lateral segregations and the degree of facilitation correlated with the relative affinity of cholesterol for the different PLs in the bilayers. Differential scanning calorimetry and (2)H nuclear magnetic resonance showed that cholesterol increased the thermostability of both the gel and lo-domains. Increased number of double bonds in the unsaturated PL increased the order in the lo-domains, likely by enriching the ordered domains in saturated lipids and cholesterol. This supported the conclusions from the trans-parinaric acid experiments, and offers insight into how cholesterol facilitated lateral segregation. In conclusion, the relative affinity of cholesterol for different PLs appears to be an important determinant for the formation of ordered domains. Our data suggests that knowledge of the affinity of cholesterol for the different PLs in a bilayer allows prediction of the degree to which the sterol promotes lo-domain formation. PMID:27508438

  9. A sol-gel-integrated protein array system for affinity analysis of aptamer-target protein interaction.

    PubMed

    Ahn, Ji-Young; Kim, Eunkyung; Kang, Jeehye; Kim, Soyoun

    2011-06-01

    A sol-gel microarray system was developed for a protein interaction assay with high activity. Comparing to 2-dimensional microarray surfaces, sol-gel can offer a more dynamic and broad range for proteins. In the present study, this sol-gel-integrated protein array was used in binding affinity analysis for aptamers. Six RNA aptamers and their target protein, yeast TBP (TATA-binding protein), were used to evaluate this method. A TBP-containing sol-gel mixture was spotted using a dispensing workstation under high-humidity conditions and each Cy-3-labeled aptamer was incubated. The dissociation constants (K(d)) were calculated by plotting the fluorescent intensity of the bound aptamers as a function of the TBP concentrations. The K(d) value of the control aptamer was found to be 8 nM, which agrees well with the values obtained using the conventional method, electric mobility shift assay. The sol-gel-based binding affinity measurements fit well with conventional binding affinity measurements, suggesting their possible use as an alternative to the conventional method. In addition, aptamer affinity measurements by the sol-gel-integrated protein chip make it possible to develop a simple high-throughput affinity method for screening high-affinity aptamers. PMID:21749295

  10. Stoichiometry and Affinity of Thioflavin T Binding to Sup35p Amyloid Fibrils

    PubMed Central

    Sulatskaya, Anna I.; Kuznetsova, Irina M.; Belousov, Mikhail V.; Bondarev, Stanislav A.; Zhouravleva, Galina A.; Turoverov, Konstantin K.

    2016-01-01

    In this work two modes of binding of the fluorescent probe thioflavin T to yeast prion protein Sup35p amyloid fibrils were revealed by absorption spectrometry of solutions prepared by equilibrium microdialysis. These binding modes exhibited significant differences in binding affinity and stoichiometry. Moreover, the absorption spectrum and the molar extinction coefficient of the dye bound in each mode were determined. The fluorescence quantum yield of the dye bound in each mode was determined via a spectrofluorimetric study of the same solutions in which the recorded fluorescence intensity was corrected for the primary inner filter effect. As previously predicted, the existence of one of the detected binding modes may be due to the incorporation of the dye into the grooves along the fiber axis perpendicular to the β-sheets of the fibrils. It was assumed that the second type of binding with higher affinity may be due to the existence of ThT binding sites that are localized to areas where amyloid fibrils are clustered. PMID:27228180

  11. Supramolecular Affinity Labeling of Histone Peptides Containing Trimethyllysine and Its Application to Histone Deacetylase Assays.

    PubMed

    Gober, Isaiah N; Waters, Marcey L

    2016-08-01

    Lysine methylation is an important histone post-translational modification (PTM) for manipulating chromatin structure and regulating gene expression, and its dysregulation is associated with various diseases including many cancers. While characterization of Lys methylation has seen improvements over the past decade due to advances in proteomic mass spectrometry and methods involving antibodies, chemical methods for selective detection of proteins containing PTMs are still lacking. Here, we detail the development of a unique labeling method wherein a synthetic receptor probe for trimethyl lysine (Kme3), CX4-ONBD, is used to direct selective fluorescent labeling of Kme3 histone peptides. This supramolecular approach reverses the paradigm of ligand-directed affinity labeling by making the receptor the synthetic component and the ligand the component to be labeled. We show that the probe mediates a strong turn-on fluorescence response in the presence of a Kme3 histone peptide and shows >5-fold selectivity in covalent labeling over an unmethylated lysine peptide. We also demonstrate the utility of the probe through the design of a turn-on fluorescence assay for histone deacetylase (HDAC) activity and for inhibitor screening and IC50 determination. Our synthetic receptor-mediated affinity labeling approach broadens the scope of PTM detection by chemical means and may facilitate the development of more versatile in vitro enzymatic assays. PMID:27387477

  12. Metalation of cyclic pseudopeptidic thiosulfinates with Ni(II) and Zn(II) after ring opening: a mechanistic investigation.

    PubMed

    Galardon, Erwan; Bourles, Emilie; Artaud, Isabelle; Daran, Jean-Claude; Roussel, Pascal; Tomas, Alain

    2007-05-28

    Thiosulfinates are an emerging class of oxidized sulfur species that are frequently supposed to be involved in biochemical processes. Reaction of 12- and 10-membered ring pseudopeptidic thiosulfinates 1a (4,4,7,7-tetramethyl-1,3,4,7,8,10-hexahydro-5,6,1,10-benzodithiadiazacyclododecine-2,9-dione 5-oxide) and 1b (3,3,6,6-tetramethyl-1,8-dihydro-4,5,1,8-benzodithiadiazecine-2,7(3H,6H)-dione 4-oxide) with a Ni(II) salt leads after ring cleavage under alkaline conditions to the isolation of diamidato/thiolato/sulfinato complexes. These two thiolato/sulfinato complexes of nickel, which can also be prepared by dioxygen oxidation of the parent diamidato/dithiolato complexes, were characterized by X-ray crystallography. They show a square-planar geometry with a S-bonded sulfinato ligand. A similar reaction between 1b and a Zn(II) salt leads to a thiolato/sulfinato complex with an O-bonded sulfinate via the intermediate formation of a mixed thiolato/sulfinic ester. On the basis of 1H NMR, IR, and mass analyses, the sulfinic ester in the intermediate is proposed to be O-bonded to the zinc center. Then, an in-depth study of the cleavage of these thiosulfinates with the oxyanions RO- and HO- was performed. This led, after trapping of the open species with CH3I, to the identification of three polyfunctionalized products containing a methyl thioether, with either an isothiazolidin-3-one S-oxide, a methyl sulfone, or a methyl sulfinic ester. All of these products arise from a selective nucleophilic attack at the sulfinyl sulfur, promoted either directly by RO- or HO- or by an internal peptidic nitrogen of the thiosulfinate after deprotonation with RO- or HO-. PMID:17465540

  13. Comparative Raman study of four plant metallothionein isoforms: Insights into their Zn(II) clusters and protein conformations.

    PubMed

    Tomas, Mireia; Tinti, Anna; Bofill, Roger; Capdevila, Mercè; Atrian, Silvia; Torreggiani, Armida

    2016-03-01

    Four Metallothioneins (MTs) from soybean (Glycine max) were heterologously synthesized and comparatively analysed by Raman spectroscopy. The participation of protein donor groups (S-thiol and N-imidazol) in Zn(II) chelation, as well as the presence of secondary structure elements was comparatively analysed. Metal clusters with different geometry can be hypothesised for the four GmMTs: a cubane-like or an adamantane-like metal cluster in Zn-GmMT1, and dinuclear Zn-S clusters in Zn-GmMT2, Zn-GmMT3 and Zn-GmMT4. The latter have also a similar average Cys/Zn content, whereas a lower ratio is present in Zn-GmMT1. This is possible thanks to the involvement in metal coordination of a greater number of bridging Cys, as well as of some carboxylate groups. As regards secondary structure elements, a large content of β-turn segments is present in all four Zn-GmMTs, especially for isoforms 1 and 4. β-strands give a contribution to the folding of three GmMTs isoforms, and the highest percentage was found in Zn-GmMT2 (~45%). Conversely, the α-helix content is negligible in all the GmMTs except in Zn-GmMT3, where this peculiar feature coincides with the possible involvement of the two His residues in metal coordination. Conversely, His is predominantly free and present as tautomer I in Zn-GmMT4. In conclusion, this work illustrates the attractive potential of Raman spectroscopy, combined with other techniques, to be a very informative tool for evidencing structural differences among in vivo synthesized metal-MT complexes. PMID:26775276

  14. Diverse Zn(II) MOFs assembled from V-shaped asymmetric multicarboxylate and N-donor ligands

    NASA Astrophysics Data System (ADS)

    Ye, Run-Ping; Yang, Jin-Xia; Zhang, Xin; Zhang, Lei; Yao, Yuan-Gen

    2016-02-01

    By reacting an asymmetry semi-rigid V-shaped linker H3L (H3L = 3-(3-carboxyphenoxy) phthalic acid) and Zn(NO3)2·6H2O under different N-donor ligands in different solvents, four new Zn-based coordination polymers, [Zn(HL)(2,2‧-bpy)(H2O)]n(1), [Zn(HL)(4,4‧-bpy)]n·n(DMA) (2), [Zn3(L)2(phen)3(H2O)]n·n(H2O) (3) and [Zn(HL)(phen)(H2O)]2(4) (2,2‧-bpy = 2,2‧-bipyridine; 4,4‧-bpy = 4,4‧-bipyridine; phen = 1,10-phenanthroline; DMA = N,N-dimethylacetamide) have been obtained. All of these compounds have been clearly identified by single crystal X-ray diffraction analysis. Compound 1 exhibits one-dimensional (1D) chain structure constructed from uninuclear Zn(II) motif, which further extends into 2D supramolecular architecture via intermolecular π-π interactions and hydrogen bonds. Structural analysis reveals that the structure of 2 and 3 can be described as a 2D hcb topology network with the point symbol of {63}. Compound 4 shows a 0D binuclear motif while its 3D packing network has a large potential solvent voids. The results of this research demonstrate that the solvent and the secondary ligands could co-regulate different structural coordination polymers with interesting properties. In addition, the thermal stabilities and solid-state luminescence properties of compounds 1-4 have also been investigated.

  15. Tripodal chelating ligand-based sensor for selective determination of Zn(II) in biological and environmental samples.

    PubMed

    Singh, Ashok Kumar; Mehtab, Sameena; Singh, Udai P; Aggarwal, Vaibhave

    2007-08-01

    Potassium hydrotris(N-tert-butyl-2-thioimidazolyl)borate [KTtt-Bu] and potassium hydrotris(3-tert-butyl-5-isopropyl-l-pyrazolyl)borate [KTpt-Bu,i-Pr] have been synthesized and evaluated as ionophores for preparation of a poly(vinyl chloride) (PVC) membrane sensor for Zn(II) ions. The effect of different plasticizers, viz. benzyl acetate (BA), dioctyl phthalate (DOP), dibutyl phthalate (DBP), tributyl phosphate (TBP), and o-nitrophenyl octyl ether (o-NPOE), and the anion excluders sodium tetraphenylborate (NaTPB), potassium tetrakis(p-chlorophenyl)borate (KTpClPB), and oleic acid (OA) were studied to improve the performance of the membrane sensor. The best performance was obtained from a sensor with a of [KTtt-Bu] membrane of composition (mg): [KTtt-Bu] (15), PVC (150), DBP (275), and NaTPB (4). This sensor had a Nernstian response (slope, 29.4+/-0.2 mV decade of activity) for Zn2+ ions over a wide concentration range (1.4x10(-7) to 1.0x10(-1) mol L(-1)) with a limit of detection of 9.5x10(-8) mol L(-1). It had a relatively fast response time (12 s) and could be used for 3 months without substantial change of the potential. The membrane sensor had very good selectivity for Zn2+ ions over a wide variety of other cations and could be used in a working pH range of 3.5-7.8. The sensor was also found to work satisfactorily in partially non-aqueous media and could be successfully used for estimation of zinc at trace levels in biological and environmental samples. PMID:17622519

  16. Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography.

    PubMed

    Li, Shangyong; Wang, Linna; Yang, Juan; Bao, Jing; Liu, Junzhong; Lin, Shengxiang; Hao, Jianhua; Sun, Mi

    2016-06-01

    In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases. PMID:27058973

  17. Removal of Ni(II), Zn(II) and Pb(II) ions from single metal aqueous solution using rice husk-based activated carbon

    SciTech Connect

    Taha, Mohd F. Shaharun, Maizatul S.; Shuib, Anis Suhaila Borhan, Azry

    2014-10-24

    An attempt was made to investigate the potential of rice husk-based activated carbon as an alternative low-cost adsorbent for the removal of Ni(II), Zn(II) and Pb(II) ions from single aqueous solution. Rice husk-based activated carbon was prepared via treatment of rice husk with NaOH followed by the carbonization process at 400°C for 2 hours. Three samples, i.e. raw rice husk, rice husk treated with NaOH and rice husk-based activated carbon, were analyzed for their morphological characteristics using field-emission scanning electron microscope/energy dispersive X-ray (FESEM/EDX). These samples were also analyzed for their carbon, hydrogen, nitrogen, oxygen and silica contents using CHN elemental analyzer and FESEM/EDX. The porous properties of rice husk-based activated carbon were determined by Brunauer-Emmett-Teller (BET) surface area analyzer, and its surface area and pore volume were 255 m{sup 2}/g and 0.17 cm{sup 2}/g, respectively. The adsorption studies for the removal of Ni(II), Zn(II) and Pb(II) ions from single metal aqueous solution were carried out at a fixed initial concentration of metal ion (150 ppm) with variation amount of adsorbent (rice husk-based activated carbon) as a function of varied contact time at room temperature. The concentration of each metal ion was analyzed using atomic absorption spectrophotometer (AAS). The results obtained from adsorption studies indicate the potential of rice husk as an economically promising precursor for the preparation of activated carbon for removal of Ni(II), Zn(II) and Pb(II) ions from single aqueous solution. Isotherm and kinetic model analyses suggested that the experimental data of adsorption studies fitted well with Langmuir, Freundlich and second-order kinetic models.

  18. Removal of Ni(II), Zn(II) and Pb(II) ions from single metal aqueous solution using rice husk-based activated carbon

    NASA Astrophysics Data System (ADS)

    Taha, Mohd F.; Shuib, Anis Suhaila; Shaharun, Maizatul S.; Borhan, Azry

    2014-10-01

    An attempt was made to investigate the potential of rice husk-based activated carbon as an alternative low-cost adsorbent for the removal of Ni(II), Zn(II) and Pb(II) ions from single aqueous solution. Rice husk-based activated carbon was prepared via treatment of rice husk with NaOH followed by the carbonization process at 400°C for 2 hours. Three samples, i.e. raw rice husk, rice husk treated with NaOH and rice husk-based activated carbon, were analyzed for their morphological characteristics using field-emission scanning electron microscope/energy dispersive X-ray (FESEM/EDX). These samples were also analyzed for their carbon, hydrogen, nitrogen, oxygen and silica contents using CHN elemental analyzer and FESEM/EDX. The porous properties of rice husk-based activated carbon were determined by Brunauer-Emmett-Teller (BET) surface area analyzer, and its surface area and pore volume were 255 m2/g and 0.17 cm2/g, respectively. The adsorption studies for the removal of Ni(II), Zn(II) and Pb(II) ions from single metal aqueous solution were carried out at a fixed initial concentration of metal ion (150 ppm) with variation amount of adsorbent (rice husk-based activated carbon) as a function of varied contact time at room temperature. The concentration of each metal ion was analyzed using atomic absorption spectrophotometer (AAS). The results obtained from adsorption studies indicate the potential of rice husk as an economically promising precursor for the preparation of activated carbon for removal of Ni(II), Zn(II) and Pb(II) ions from single aqueous solution. Isotherm and kinetic model analyses suggested that the experimental data of adsorption studies fitted well with Langmuir, Freundlich and second-order kinetic models.

  19. Thirty-fifth anniversary of the optical affinity sensor for glucose: a personal retrospective.

    PubMed

    Schultz, Jerome S

    2015-01-01

    Since 1962 when Clark introduced the enzyme electrode, research has been intense for a robust implantable glucose sensor. An alternative "optical affinity sensor" was introduced by Jerome Schultz in 1979. The evolution of this sensor technology into a new methodology is reviewed. The approach integrates a variety of disparate concepts: the selectivity of immunoassays-selectivity for glucose was obtained with concanavalin A, detection sensitivity was obtained with fluorescence (FITC-Dextran), and miniaturization was achieved by the use of an optical fiber readout system. Refinements of Schultz's optical affinity sensor approach over the past 35 years have led to a number of configurations that show great promise to meet the needs of a successful implantable continuous monitoring device for diabetics, some of which are currently being tested clinically. PMID:25269660

  20. Structure-based Design of Peptides with High Affinity and Specificity to HER2 Positive Tumors

    PubMed Central

    Geng, Lingling; Wang, Zihua; Yang, Xiaoliang; Li, Dan; Lian, Wenxi; Xiang, Zhichu; Wang, Weizhi; Bu, Xiangli; Lai, Wenjia; Hu, Zhiyuan; Fang, Qiaojun

    2015-01-01

    To identify peptides with high affinity and specificity against human epidermal growth factor receptor 2 (HER2), a series of peptides were designed based on the structure of HER2 and its Z(HER2:342) affibody. By using a combination protocol of molecular dynamics modeling, MM/GBSA binding free energy calculations, and binding free energy decomposition analysis, two novel peptides with 27 residues, pep27 and pep27-24M, were successfully obtained. Immunocytochemistry and flow cytometry analysis verified that both peptides can specifically bind to the extracellular domain of HER2 protein at cellular level. The Surface Plasmon Resonance imaging (SPRi) analysis showed that dissociation constants (KD) of these two peptides were around 300 nmol/L. Furthermore, fluorescence imaging of peptides against nude mice xenografted with SKBR3 cells indicated that both peptides have strong affinity and high specificity to HER2 positive tumors. PMID:26284145

  1. Structure-based Design of Peptides with High Affinity and Specificity to HER2 Positive Tumors.

    PubMed

    Geng, Lingling; Wang, Zihua; Yang, Xiaoliang; Li, Dan; Lian, Wenxi; Xiang, Zhichu; Wang, Weizhi; Bu, Xiangli; Lai, Wenjia; Hu, Zhiyuan; Fang, Qiaojun

    2015-01-01

    To identify peptides with high affinity and specificity against human epidermal growth factor receptor 2 (HER2), a series of peptides were designed based on the structure of HER2 and its Z(HER2:342) affibody. By using a combination protocol of molecular dynamics modeling, MM/GBSA binding free energy calculations, and binding free energy decomposition analysis, two novel peptides with 27 residues, pep27 and pep27-24M, were successfully obtained. Immunocytochemistry and flow cytometry analysis verified that both peptides can specifically bind to the extracellular domain of HER2 protein at cellular level. The Surface Plasmon Resonance imaging (SPRi) analysis showed that dissociation constants (K D) of these two peptides were around 300 nmol/L. Furthermore, fluorescence imaging of peptides against nude mice xenografted with SKBR3 cells indicated that both peptides have strong affinity and high specificity to HER2 positive tumors. PMID:26284145

  2. Effect of biofilm coatings at metal-oxide/water interfaces II: Competitive sorption between Pb(II) and Zn(II) at Shewanella oneidensis/metal-oxide/water interfaces

    NASA Astrophysics Data System (ADS)

    Wang, Yingge; Gélabert, Alexandre; Michel, F. Marc; Choi, Yongseong; Eng, Peter J.; Spormann, Alfred M.; Brown, Gordon E.

    2016-09-01

    Competitive sorption of Pb(II) and Zn(II) on Shewanella oneidensis MR-1 biofilm-coated single-crystal α-Al2O3 (1 -1 0 2) and α-Fe2O3 (0 0 0 1) surfaces was investigated using long-period X-ray standing wave-florescence yield (LP-XSW-FY) spectroscopy. In situ partitioning of aqueous Pb(II) and Zn(II) between the biofilms and underlying metal-oxide substrates was probed following exposure of these complex interfaces to equi-molar Pb and Zn solutions (0.01 M NaNO3 as background electrolyte, pH = 6.0, and 3-h equilibration time). At higher Pb and Zn concentrations (⩾10-5 M), more than 99% of these ions partitioned into the biofilms at S. oneidensis/α-Al2O3 (1 -1 0 2)/water interfaces, which is consistent with the partitioning behavior of both Pb(II) or Zn(II) in single-metal-ion experiments. Thus, no apparent competitive effects were found in this system at these relatively high metal-ion concentrations. However, at lower equi-molar concentrations (⩽10-6 M), Pb(II) and Zn(II) partitioning in the same system changed significantly compared to the single-metal-ion systems. The presence of Zn(II) decreased Pb(II) partitioning onto α-Al2O3 (1 -1 0 2) substantially (∼52% to ∼13% at 10-7 M, and ∼23% to ∼5% at 10-6 M), whereas the presence of Pb(II) caused more Zn(II) to partition onto α-Al2O3 (1 -1 0 2) surfaces (∼15% to ∼28% at 10-7 M, and ∼1% to ∼7% at 10-6 M). The higher observed partitioning of Zn(II) (∼28%) at the α-Al2O3 (1 -1 0 2) surfaces compared to Pb(II) (∼13%) in the mixed-metal-ion systems at the lowest concentration (10-7 M) suggests that Zn(II) is slightly favored over Pb(II) for sorption sites on α-Al2O3 (1 -1 0 2) surfaces under our experimental conditions. Competitive sorption of Pb(II) and Zn(II) at S. oneidensis/α-Fe2O3 (0 0 0 1)/water interfaces at equi-molar metal-ion concentrations of ⩽10-6 M showed that the presence of Pb(II) ions decreased Zn(II) partitioning onto α-Fe2O3 (0 0 0 1) significantly (∼45% to <1% at 10

  3. Synthesis, spectroscopic characterization and biological activities of N4O2 Schiff base ligand and its metal complexes of Co(II), Ni(II), Cu(II) and Zn(II)

    NASA Astrophysics Data System (ADS)

    Al-Resayes, Saud I.; Shakir, Mohammad; Abbasi, Ambreen; Amin, Kr. Mohammad Yusuf; Lateef, Abdul

    The Schiff base ligand, bis(indoline-2-one)triethylenetetramine (L) obtained from condensation of triethylenetetramine and isatin was used to synthesize the complexes of type, [ML]Cl2 [M = Co(II), Ni(II), Cu(II) and Zn(II)]. L was characterized on the basis of the results of elemental analysis, FT-IR, 1H and 13C NMR, mass spectroscopic studies. The stoichiometry, bonding and stereochemistries of complexes were ascertained on the basis of results of elemental analysis, magnetic susceptibility values, molar conductance and various spectroscopic studies. EPR, UV-vis and magnetic moments revealed an octahedral geometry for complexes. L and its Cu(II) and Zn(II) complexes were screened for their antibacterial activity. Analgesic activity of Cu(II) and Zn(II) complexes was also tested in rats by tail flick method. Both complexes were found to possess good antibacterial and moderate analgesic activity.

  4. Synthesis and theoretical study of 5-methoxyisatin-3-(N-cyclohexyl)thiosemicarbazone and its Ni(II) and Zn(II) complexes

    NASA Astrophysics Data System (ADS)

    Kandemirli, Fatma; Arslan, Taner; Karadayı, Nevzat; Ebenso, Eno. E.; Köksoy, Baybars

    2009-12-01

    5-Methoxyisatin-3-(N-cyclohexyl)thiosemicarbazone (H 2MICT) and its Zn(II) and Ni(II) complexes have been synthesized and characterized using IR, 1H NMR, 13C-NMR, MS, UV and elemental analysis. (H 2MICT) ligand has been characterized with X-ray diffraction method also. The possible structures and IR data of the studied molecules were calculated and compared with experimental results using B3LYP/6-31G(d,p) and B3LYP/LANL2DZ methods.

  5. Thermal studies and vibrational analyses of m-methylaniline complexes of Zn(II), Cd(II) and Hg(II) bromides

    NASA Astrophysics Data System (ADS)

    Golcuk, K.; Altun, A.; Kumru, M.

    2003-06-01

    The complexes having the MBr2L2 (M: Zn, Cd and Hg; L: m-methylaniline) formulae have been prepared and characterized by their elemental analyses, thermogravimetric analyses, IR and Raman spectral studies. IR and Raman bands of the complexes have been assigned as compared with the free ligand. Coordination effects on the internal modes of m-methylaniline have been discussed. Vibrational spectra propose that the [ZnBr2(mMA)2] complex is in a tetrahedral environment around Zn(II) ion with C2v symmetry whereas Cd(II) and Hg(II) complexes have 5-coordinate polymeric bromide bridged structures.

  6. Thermal Studies of Zn(II), Cd(II) and Hg(II) Complexes of Some N-Alkyl-N-Phenyl-Dithiocarbamates

    PubMed Central

    Onwudiwe, Damian C.; Ajibade, Peter A.

    2012-01-01

    The thermal decomposition of Zn(II), Cd(II) and Hg(II) complexes of N-ethyl-N-phenyl and N-butyl-N-phenyl dithiocarbamates have been studied using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The products of the decomposition, at two different temperatures, were further characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). The results show that while the zinc and cadmium complexes undergo decomposition to form metal sulphides, and further undergo oxidation forming metal oxides as final products, the mercury complexes gave unstable volatiles as the final product. PMID:22949811

  7. Smooth big bounce from affine quantization

    NASA Astrophysics Data System (ADS)

    Bergeron, Hervé; Dapor, Andrea; Gazeau, Jean Pierre; Małkiewicz, Przemysław

    2014-04-01

    We examine the possibility of dealing with gravitational singularities on a quantum level through the use of coherent state or wavelet quantization instead of canonical quantization. We consider the Robertson-Walker metric coupled to a perfect fluid. It is the simplest model of a gravitational collapse, and the results obtained here may serve as a useful starting point for more complex investigations in the future. We follow a quantization procedure based on affine coherent states or wavelets built from the unitary irreducible representation of the affine group of the real line with positive dilation. The main issue of our approach is the appearance of a quantum centrifugal potential allowing for regularization of the singularity, essential self-adjointness of the Hamiltonian, and unambiguous quantum dynamical evolution.

  8. Improved native affinity purification of RNA.

    PubMed

    Batey, Robert T; Kieft, Jeffrey S

    2007-08-01

    RNA biochemical or structural studies often require an RNA sample that is chemically pure, and most protocols for its in vitro production use denaturing polyacrylamide gel electrophoresis to achieve this. Unfortunately, many RNAs do not quantitatively refold into an active conformation after denaturation, creating significant problems for downstream characterization or use. In addition, this traditional purification method is not amenable to studies demanding high-throughput RNA production. Recently, we presented the first general method for producing almost any RNA sequence that employs an affinity tag that is removed during the purification process. Because technical difficulties prevented application of this method to many RNAs, we have developed an improved version that utilizes a different activatable ribozyme and affinity tag that are considerably more robust, rapid, and broadly applicable. PMID:17548432

  9. Protein affinity map of chemical space.

    PubMed

    Kauvar, L M; Villar, H O; Sportsman, J R; Higgins, D L; Schmidt, D E

    1998-09-11

    Affinity fingerprinting is a quantitative method for mapping chemical space based on binding preferences of compounds for a reference panel of proteins. An effective reference panel of <20 proteins can be empirically selected which shows differential interaction with nearly all compounds. By using this map to iteratively sample the chemical space, identification of active ligands from a library of 30,000 candidate compounds has been accomplished for a wide spectrum of specific protein targets. In each case, <200 compounds were directly assayed against the target. Further, analysis of the fingerprint database suggests a strategy for effective selection of affinity chromatography ligands and scaffolds for combinatorial chemistry. With such a system, the large numbers of potential therapeutic targets emerging from genome research can be categorized according to ligand binding properties, complementing sequence based classification. PMID:9792501

  10. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  11. A Fluorescence Lecture Demonstration.

    ERIC Educational Resources Information Center

    Bozzelli, Joseph W.; Kemp, Marwin

    1982-01-01

    Describes fluorescence demonstrations related to several aspects of molecular theory and quantitized energy levels. Demonstrations use fluorescent chemical solutions having luminescence properties spanning the visible spectrum. Also describes a demonstration of spontaneous combustion of familiar substances in chlorine. (JN)

  12. Fluorescent optical position sensor

    DOEpatents

    Weiss, Jonathan D.

    2005-11-15

    A fluorescent optical position sensor and method of operation. A small excitation source side-pumps a localized region of fluorescence at an unknown position along a fluorescent waveguide. As the fluorescent light travels down the waveguide, the intensity of fluorescent light decreases due to absorption. By measuring with one (or two) photodetectors the attenuated intensity of fluorescent light emitted from one (or both) ends of the waveguide, the position of the excitation source relative to the waveguide can be determined by comparing the measured light intensity to a calibrated response curve or mathematical model. Alternatively, excitation light can be pumped into an end of the waveguide, which generates an exponentially-decaying continuous source of fluorescent light along the length of the waveguide. The position of a photodetector oriented to view the side of the waveguide can be uniquely determined by measuring the intensity of the fluorescent light emitted radially at that location.

  13. Evaluation of Chemical Fluorescent Dyes as a Protein Conjugation Partner for Live Cell Imaging

    PubMed Central

    Hayashi-Takanaka, Yoko; Stasevich, Timothy J.; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

    2014-01-01

    To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye∶protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red). PMID:25184362

  14. A MEMS Dielectric Affinity Glucose Biosensor.

    PubMed

    Huang, Xian; Li, Siqi; Davis, Erin; Li, Dachao; Wang, Qian; Lin, Qiao

    2013-06-20

    Continuous glucose monitoring (CGM) sensors based on affinity detection are desirable for long-term and stable glucose management. However, most affinity sensors contain mechanical moving structures and complex design in sensor actuation and signal readout, limiting their reliability in subcutaneously implantable glucose detection. We have previously demonstrated a proof-of-concept dielectric glucose sensor that measured pre-mixed glucose-sensitive polymer solutions at various glucose concentrations. This sensor features simplicity in sensor design, and possesses high specificity and accuracy in glucose detection. However, lack of glucose diffusion passage, this device is unable to fulfill real-time in-vivo monitoring. As a major improvement to this device, we present in this paper a fully implantable MEMS dielectric affinity glucose biosensor that contains a perforated electrode embedded in a suspended diaphragm. This capacitive-based sensor contains no moving parts, and enables glucose diffusion and real-time monitoring. The experimental results indicate that this sensor can detect glucose solutions at physiological concentrations and possesses good reversibility and reliability. This sensor has a time constant to glucose concentration change at approximately 3 min, which is comparable to commercial systems. The sensor has potential applications in fully implantable CGM that require excellent long-term stability and reliability. PMID:24511215

  15. On constructing purely affine theories with matter

    NASA Astrophysics Data System (ADS)

    Cervantes-Cota, Jorge L.; Liebscher, D.-E.

    2016-08-01

    We explore ways to obtain the very existence of a space-time metric from an action principle that does not refer to it a priori. Although there are reasons to believe that only a non-local theory can viably achieve this goal, we investigate here local theories that start with Schrödinger's purely affine theory (Schrödinger in Space-time structure. Cambridge UP, Cambridge, 1950), where he gave reasons to set the metric proportional to the Ricci curvature aposteriori. When we leave the context of unified field theory, and we couple the non-gravitational matter using some weak equivalence principle, we can show that the propagation of shock waves does not define a lightcone when the purely affine theory is local and avoids the explicit use of the Ricci tensor in realizing the weak equivalence principle. When the Ricci tensor is substituted for the metric, the equations seem to have only a very limited set of solutions. This backs the conviction that viable purely affine theories have to be non-local.

  16. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture. PMID:26584922

  17. Safe biodegradable fluorescent particles

    DOEpatents

    Martin, Sue I.; Fergenson, David P.; Srivastava, Abneesh; Bogan, Michael J.; Riot, Vincent J.; Frank, Matthias

    2010-08-24

    A human-safe fluorescence particle that can be used for fluorescence detection instruments or act as a safe simulant for mimicking the fluorescence properties of microorganisms. The particle comprises a non-biological carrier and natural fluorophores encapsulated in the non-biological carrier. By doping biodegradable-polymer drug delivery microspheres with natural or synthetic fluorophores, the desired fluorescence can be attained or biological organisms can be simulated without the associated risks and logistical difficulties of live microorganisms.

  18. Atmospheric Nitrogen Fluorescence Yield

    NASA Technical Reports Server (NTRS)

    Adams, J. H., Jr.; Christl, M. J.; Fountain, W. F.; Gregory, J. C.; Martens, K. U.; Sokolsky, Pierre; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    Several existing and planned experiments estimate the energies of ultra-high energy cosmic rays from air showers using the atmospheric nitrogen fluorescence. The nitrogen fluorescence yield from air shower electrons depends on the atmospheric composition. We will discuss the uncertainties in the fluorescence yield form electrons in the real atmosphere and describe a concept for a small balloon payload to measure the atmospheric fluorescence yield as a function of attitude.

  19. Mining the soluble chloroplast proteome by affinity chromatography.

    PubMed

    Bayer, Roman G; Stael, Simon; Csaszar, Edina; Teige, Markus

    2011-04-01

    Chloroplasts are fundamental organelles enabling plant photoautotrophy. Besides their outstanding physiological role in fixation of atmospheric CO(2), they harbor many important metabolic processes such as biosynthesis of amino acids, vitamins or hormones. Technical advances in MS allowed the recent identification of most chloroplast proteins. However, for a deeper understanding of chloroplast function it is important to obtain a complete list of constituents, which is so far limited by the detection of low-abundant proteins. Therefore, we developed a two-step strategy for the enrichment of low-abundant soluble chloroplast proteins from Pisum sativum and their subsequent identification by MS. First, chloroplast protein extracts were depleted from the most abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase by SEC or heating. Further purification was carried out by affinity chromatography, using ligands specific for ATP- or metal-binding proteins. By these means, we were able to identify a total of 448 proteins including 43 putative novel chloroplast proteins. Additionally, the chloroplast localization of 13 selected proteins was confirmed using yellow fluorescent protein fusion analyses. The selected proteins included a phosphoglycerate mutase, a cysteine protease, a putative protein kinase and an EF-hand containing substrate carrier protein, which are expected to exhibit important metabolic or regulatory functions. PMID:21365755

  20. Affinity entrapment of oligosaccharides and glycopeptides using free lectin solution.

    PubMed

    Yodoshi, Masahiro; Oyama, Takehiro; Masaki, Ken; Kakehi, Kazuaki; Hayakawa, Takao; Suzuki, Shigeo

    2011-01-01

    Two procedures were proposed for the specific recovery of fluorescent derivatives of glycoprotein-derived oligosaccharides and tryptic glycopeptides using certain plant lectins. The first was based on the salting out of oligosaccharide-lectin conjugates with ammonium sulfate. Oligosaccharides specifically bound to lectins were recovered free from lectins using ethanol precipitation after dissolution in water. This method enabled group separation of 2-aminopyridine-labeled oligosaccharides derived from ovalbumin to galacto-oligosaccharides and agalacto-oligosaccharides by Ricinus communis agglutinin, and to high mannose- and hybrid-type oligosaccharides by wheat-germ agglutinin. Fractional precipitation based on differences in affinity for concanavalin A was accomplished by adding an appropriate concentration of methyl α-mannoside as an inhibitor. In the second method, tryptic digests of glycoproteins were mixed with a lectin solution, and the glycopeptide-lectin conjugates were specifically trapped on a centrifugal ultrafiltration membrane with cut-off of 10 kD. Trapped glycopeptides, as retentates, were passed through membranes by resuspension in diluted acid. This method is particularly useful for the enrichment of glycopeptides in protease digestion mixtures for glycosylation analyses by liquid chromatography-mass spectrometry. PMID:21478615

  1. Mining the soluble chloroplast proteome by affinity chromatography

    PubMed Central

    Bayer, Roman G; Stael, Simon; Csaszar, Edina; Teige, Markus

    2011-01-01

    Chloroplasts are fundamental organelles enabling plant photoautotrophy. Besides their outstanding physiological role in fixation of atmospheric CO2, they harbor many important metabolic processes such as biosynthesis of amino acids, vitamins or hormones. Technical advances in MS allowed the recent identification of most chloroplast proteins. However, for a deeper understanding of chloroplast function it is important to obtain a complete list of constituents, which is so far limited by the detection of low-abundant proteins. Therefore, we developed a two-step strategy for the enrichment of low-abundant soluble chloroplast proteins from Pisum sativum and their subsequent identification by MS. First, chloroplast protein extracts were depleted from the most abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase by SEC or heating. Further purification was carried out by affinity chromatography, using ligands specific for ATP- or metal-binding proteins. By these means, we were able to identify a total of 448 proteins including 43 putative novel chloroplast proteins. Additionally, the chloroplast localization of 13 selected proteins was confirmed using yellow fluorescent protein fusion analyses. The selected proteins included a phosphoglycerate mutase, a cysteine protease, a putative protein kinase and an EF-hand containing substrate carrier protein, which are expected to exhibit important metabolic or regulatory functions. PMID:21365755

  2. Methods for determining the genetic affinity of microorganisms and viruses

    NASA Technical Reports Server (NTRS)

    Fox, George E. (Inventor); Willson, III, Richard C. (Inventor); Zhang, Zhengdong (Inventor)

    2012-01-01

    Selecting which sub-sequences in a database of nucleic acid such as 16S rRNA are highly characteristic of particular groupings of bacteria, microorganisms, fungi, etc. on a substantially phylogenetic tree. Also applicable to viruses comprising viral genomic RNA or DNA. A catalogue of highly characteristic sequences identified by this method is assembled to establish the genetic identity of an unknown organism. The characteristic sequences are used to design nucleic acid hybridization probes that include the characteristic sequence or its complement, or are derived from one or more characteristic sequences. A plurality of these characteristic sequences is used in hybridization to determine the phylogenetic tree position of the organism(s) in a sample. Those target organisms represented in the original sequence database and sufficient characteristic sequences can identify to the species or subspecies level. Oligonucleotide arrays of many probes are especially preferred. A hybridization signal can comprise fluorescence, chemiluminescence, or isotopic labeling, etc.; or sequences in a sample can be detected by direct means, e.g. mass spectrometry. The method's characteristic sequences can also be used to design specific PCR primers. The method uniquely identifies the phylogenetic affinity of an unknown organism without requiring prior knowledge of what is present in the sample. Even if the organism has not been previously encountered, the method still provides useful information about which phylogenetic tree bifurcation nodes encompass the organism.

  3. Localization of Free Field Realizations of Affine Lie Algebras

    NASA Astrophysics Data System (ADS)

    Futorny, Vyacheslav; Grantcharov, Dimitar; Martins, Renato A.

    2015-04-01

    We use localization technique to construct new families of irreducible modules of affine Kac-Moody algebras. In particular, localization is applied to the first free field realization of the affine Lie algebra or, equivalently, to imaginary Verma modules.

  4. Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery.

    PubMed

    Lo, Mei-Chu; Aulabaugh, Ann; Jin, Guixian; Cowling, Rebecca; Bard, Jonathan; Malamas, Michael; Ellestad, George

    2004-09-01

    The fluorescence-based thermal shift assay is a general method for identification of inhibitors of target proteins from compound libraries. Using an environmentally sensitive fluorescent dye to monitor protein thermal unfolding, the ligand-binding affinity can be assessed from the shift of the unfolding temperature (Delta Tm) obtained in the presence of ligands relative to that obtained in the absence of ligands. In this article, we report that the thermal shift assay can be conducted in an inexpensive, commercially available device for temperature control and fluorescence detection. The binding affinities obtained from thermal shift assays are compared with the binding affinities measured by isothermal titration calorimetry and with the IC(50) values from enzymatic assays. The potential pitfalls in the data analysis of thermal shift assays are also discussed. PMID:15301960

  5. Shape-selective sensing of lipids in aqueous solution by a designed fluorescent molecular tube.

    PubMed

    Shorthill, Berkeley J; Avetta, Christopher T; Glass, Timothy E

    2004-10-13

    The design, synthesis, and recognition properties of two isomeric calixnaphthalene-type molecular tubes is described. The anti isomer showed strong fluorescence quenching upon titration with various simple lipid guests in water, in which the more hydrophobic guests associated with higher affinity. The syn isomer bound long straight-chain guests tightly with a similar fluorescence-quenching response. However, the syn isomer was more selective, giving an increase in fluorescence upon titration of guests which could not thread through the tube. Thus, the syn isomer is a selective fluorescent sensor for long straight-chain lipids. PMID:15469241

  6. Novel Chalcone-Based Fluorescent Human Histamine H3 Receptor Ligands as Pharmacological Tools

    PubMed Central

    Tomasch, Miriam; Schwed, J. Stephan; Weizel, Lilia; Stark, Holger

    2012-01-01

    Novel fluorescent chalcone-based ligands at human histamine H3 receptors (hH3R) have been designed, synthesized, and characterized. Compounds described are non-imidazole analogs of ciproxifan with a tetralone motif. Tetralones as chemical precursors and related fluorescent chalcones exhibit affinities at hH3R in the same concentration range like the reference antagonist ciproxifan (hH3R pKi value of 7.2). Fluorescence characterization of our novel ligands shows emission maxima about 570 nm for yellow fluorescent chalcones and ≥600 nm for the red fluorescent derivatives. Interferences to cellular autofluorescence could be excluded. All synthesized chalcone compounds could be used to visualize hH3R proteins in stably transfected HEK-293 cells using confocal laser scanning fluorescence microscopy. These novel fluorescent ligands possess high potential to be used as pharmacological tools for hH3R visualization in different tissues. PMID:22470321

  7. Fluorescence study of sugars

    NASA Astrophysics Data System (ADS)

    Thongjamroon, Sunida; Pattanaporkratana, Apichart

    2015-07-01

    We studied photoemission of monosaccharides and disaccharides using laser-induced fluorescence spectroscopy. A 532- nm, 10 mW, laser was used to excite the samples and back-scattering signals were collected by a spectrometer. We found that most sugars show weak fluorescence in solid phase but do not fluoresce when dissolved in water solutions. The emission spectra show similar peak intensity at 590 nm, but they are different in emission intensities. We suggest that the fluorescence spectra may be used to differentiate sugar type, even though the origin of the fluorescence is unclear and needed further study.

  8. Affinity maturation of single-chain variable fragment specific for aflatoxin B(1) using yeast surface display.

    PubMed

    Min, Won-Ki; Kim, Sung-Gun; Seo, Jin-Ho

    2015-12-01

    As aflatoxin B1 is one of the most toxic mycotoxins, it is important to detect and to quantify aflatoxin B1 accurately by immunological methods. To enhance aflatoxin B1-binding affinity of the single-chain variable fragment, yeast surface display technique combined with fluorescence-activated cell sorting was applied. A randomly mutated scFv library was subjected to 4 rounds of fluorescence-activated cell sorting, resulting in isolation of 5 scFv variants showing an affinity improvement compared to the parental wild type scFv. The best scFv with a 9-fold improvement in affinity for aflatoxin B1 exhibited similar specificity to the monoclonal antibody. Most of the mutations in scFv-M37 were located outside of the canonical antigen-contact loops, suggesting that its affinity improvement might be driven by an allosteric effect inducing scFv-M37 to form a more favorable binding pocket for aflatoxin B1 than the wild type scFv. PMID:26041237

  9. Binding studies of tear lipocalin: the role of the conserved tryptophan in maintaining structure, stability and ligand affinity.

    PubMed

    Gasymov, O K; Abduragimov, A R; Yusifov, T N; Glasgow, B J

    1999-08-17

    The principal lipid binding protein in tears, tear lipocalin (TL), binds acid and the fluorescent fatty acid analogs, DAUDA and 16-AP at one site TL compete for this binding site. A fluorescent competitive binding assay revealed that apo-TL has a high affinity for phospholipids and stearic acid (Ki) of 1.2 microM and 1.3 microM, respectively, and much less affinity for cholesterol (Ki) of 15.9 of the hydrocarbon chain. TL binds most strongly the least soluble lipids permitting these lipids to exceed their maximum solubility in aqueous solution. These data implicate TL in solubilizing and transporting lipids in the tear film. Phenylalanine, tyrosine and cysteine+ were substituted for TRP 17, the only invariant residue throughout the lipocalin superfamily. Cysteine substitution resulted in some loss os secondary structure, relaxation of aromatic side chain rigidity, decreased binding affinity for DAUDA and destabilization of structure. Mutants of TL, W17Y, and W17F showed a higher binding affinity for DAUDA than wild-type TL. Comparison of the results of the tryptophan 17 substitution in lipocalin with those of tryptophan 19 substitution in beta-lactoglobulin revealed important differences in binding characteristics that reflect the functional heterogeneity within the lipocalin family. PMID:10515687

  10. Fluorescent probes for G-quadruplex structures.

    PubMed

    Vummidi, Balayeshwanth R; Alzeer, Jawad; Luedtke, Nathan W

    2013-03-18

    Mounting evidence supports the presence of biologically relevant G-quadruplexes in single-cell organisms, but the existence of endogenous G-quadruplex structures in mammalian cells remains highly controversial. This is due, in part, to the common misconception that DNA and RNA molecules are passive information carriers with relatively little structural or functional complexity. For those working in the field, however, the lack of available tools for characterizing DNA structures in vivo remains a major limitation to addressing fundamental questions about structure-function relationships of nucleic acids. In this review, we present progress towards the direct detection of G-quadruplex structures by using small molecules and modified oligonucleotides as fluorescent probes. While most development has focused on cell-permeable probes that selectively bind to G-quadruplex structures with high affinity, these same probes can induce G-quadruplex folding, thereby making the native conformation of the DNA or RNA molecule (i.e., in the absence of probe) uncertain. For this reason, modified oligonucleotides and fluorescent base analogues that serve as "internal" fluorescent probes are presented as an orthogonal means for detecting conformational changes, without necessarily perturbing the equilibria between G-quadruplex, single-stranded, and duplex DNA. The major challenges and motivation for the development of fluorescent probes for G-quadruplex structures are presented, along with a summary of the key photophysical, biophysical, and biological properties of reported examples. PMID:23440895

  11. Fluorescent indicator dyes for calcium ions

    NASA Technical Reports Server (NTRS)

    Tsien, Roger Y. (Inventor); Grynkiewicz, Grzegorz (Inventor)

    1986-01-01

    The present invention discloses a new class of highly fluorescent indicator dyes that are specific for calcium ions. The new fluorescent indicator dyes combine a stilbene-type fluorophore with a tetracarboxylate parent Ca.sup.2+ chelating compound having the octacoordinate pattern of liganding groups characteristic of EGTA and BAPTA. Preferred forms contain extra heterocyclic bridges to reinforce the ethylenic bond of the stilbene and to reduce hydrophobicity. Compared to their widely used predecessor, quin2, the new dyes offer up to thirty-fold brighter fluorescence, major changes in wavelength (not just intensity) upon Ca.sup.2+ binding, slightly lower affinities for Ca.sup.2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca.sup.2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca.sup.2+, make the dyes useful indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues. The present invention also discloses an improved method for synthesizing alpha-acyloxyalkyl bromides wherein the bromides so synthesized are free of contaminating bis(1-bromoalkyl)ether. The improved method is exemplified herein in the synthesis of acetoxymethyl bromide, a compound useful in preparing the acetoxymethyl esters disclosed herein as novel Ca.sup.2+ specific fluorescent indicators.

  12. Generation of Recombinant Antibodies to Rat GABAA Receptor Subunits by Affinity Selection on Synthetic Peptides

    PubMed Central

    Koduvayur, Sujatha P.; Gussin, Hélène A.; Parthasarathy, Rajni; Hao, Zengping; Kay, Brian K.; Pepperberg, David R.

    2014-01-01

    The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure. PMID:24586298

  13. Affinity of rosmarinic acid to human serum albumin and its effect on protein conformation stability.

    PubMed

    Peng, Xin; Wang, Xiangchao; Qi, Wei; Su, Rongxin; He, Zhimin

    2016-02-01

    Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. The interaction between RA and human serum albumin (HSA) was investigated by multi-spectroscopic, electrochemistry, molecular docking and molecular dynamics simulation methods. The fluorescence emission of HSA was quenched by RA through a combined static and dynamic quenching mechanism, but the static quenching was the major constituent. Fluorescence experiments suggested that RA was bound to HSA with moderately strong binding affinity through hydrophobic interaction. The probable binding location of RA was located near site I of HSA. Additionally, as shown by the Fourier transform infrared (FT-IR) and circular dichroism (CD) spectra, RA can result in conformational and structural alterations of HSA. Furthermore, the molecular dynamics studies were used to investigate the stability of the HSA and HSA-RA system. Altogether, the results can provide an important insight for the applications of RA in the food industry. PMID:26304336

  14. Time-Resolved Transient Optical Absorption Study of Bis(terpyridyl)oligothiophenes and Their Metallo-Supramolecular Polymers with Zn(II) Ion Couplers.

    PubMed

    Rais, David; Menšík, Miroslav; Štenclová-Bláhová, Pavla; Svoboda, Jan; Vohlídal, Jiří; Pfleger, Jiří

    2015-06-18

    α,ω-Bis(terpyridyl)oligothiophenes spontaneously assemble with Zn(II) ions giving conjugated constitutional dynamic polymers (dynamers) of the metallo-supramolecular class, which potentially might be utilized in optoelectronics. Their photophysical properties, which are of great importance in this field of application, are strongly influenced by the dynamic morphology. It was assessed in this study by using ultrafast pump-probe optical absorption spectroscopy. We identified and characterized relaxation processes running in photoexcited molecules of these oligomers and dynamers and show impacts of disturbed coplanarity of adjacent rings (twisting the thiophene-thiophene and thiophene-terpyridyl bonds by attached hexyl side groups) and Zn(II) ion couplers on these processes. Major effects are seen in the time constants of rotational relaxation, intersystem crossing, and de-excitation lifetimes. The photoexcited states formed on different repeating units within the same dynamer chain do not interact with each other even at very high excitation density. The method is presented that allows determining the equilibrium fraction of unbound oligothiophene species in a dynamer solution, from which otherwise hardly accessible values of the average degree of polymerization of constitutionally dynamic chains in solution can be estimated. PMID:25913085

  15. Versatile Tailoring of Paddle-Wheel Zn(II) Metal-Organic Frameworks through Single-Crystal-to-Single-Crystal Transformations.

    PubMed

    Pal, Tapan K; Neogi, Subhadip; Bharadwaj, Parimal K

    2015-11-01

    A new tetracarboxylate ligand having short and long arms formed 2D layer Zn(II) coordination polymer 1 with paddle-wheel secondary building units under solvothermal conditions. The framework undergoes solvent-specific single crystal-to-single crystal (SC-SC) transmetalation to produce 1Cu . With a sterically encumbered dipyridyl linker, the same ligand forms non-interpenetrated, 3D, pillared-layer Zn(II) metal-organic framework (MOF) 2, which takes part in SC-SC linker-exchange reactions to produce three daughter frameworks. The parent MOF 2 shows preferential incorporation of the longest linker in competitive linker-exchange experiments. All the 3D MOFs undergo complete SC-SC transmetalation with Cu(II) , whereby metal exchange in different solvents and monitoring of X-ray structures revealed that bulky solvated metal ions lead to ordering of the shortest linker in the framework, which confirms that the solvated metal ions enter through the pores along the linker axis. PMID:26383591

  16. Synthesis, characterization and biological activity of new mixed ligand complexes of Zn(II) naproxen with nitrogen based ligands.

    PubMed

    Abu Ali, Hijazi; Fares, Hadeel; Darawsheh, Mohanad; Rappocciolo, Emilia; Akkawi, Mutaz; Jaber, Suhair

    2015-01-01

    A series of novel Zn(II) complexes [Zn2(nap)4] (1), [Zn(nap)21,10-phen](2), [Zn(nap)22,9-dmphen] (3), [Zn(nap)2(2-ampy)2] (4), [Zn(nap)2(imid)2] (5), [Zn(nap)2(1,2-dmimid)2] (6) (nap = naproxen, 1,10-phen = 1,10-phenanthroline, 2,9-dmphen = 2,9-dimethyl-1,10-phenanthroline, 2-ampy = 2-aminopyridine, imid = imidazole, 1,2-dmimid = 1,2-dimethyl imidazole) were synthesized and characterized using IR, UV-Vis, (1)H NMR, (13)C{(1)H} NMR spectroscopy. The crystal structure of complex 3 was determined using single-crystal X-ray diffraction. In order to assess the effect of the metal ions on the anti-bacterial activity, complexes 1-6 have been screened in vitro, against (G(+)) bacteria (Staphylococcus aureus and Micrococcus luteus) and (G(-)) bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis and Escherichia coli) using the agar well diffusion method. Complex 2 was the only complex that showed antibacterial activity against P. aeruginosa, where the complexation of the parent ligand 1,10-phenathroline enhanced significantly the activity. All the complexes showed different activity against the different bacteria, and were compared with activity of the parent ligands. The complexes were tested also for their anti-malarial activity using two methods: a semi-quantitative micro-assay and a previously self-developed quantitative in-vitro method. Both were used to study the efficiency of these complexes in inhibiting the formation of the Malaria pigment. This is considered an important target of many known anti-malarial drugs such as Chloroquine and Amodaquine. Results showed that the efficiency of complex 3 in preventing the formation of β-hematin was 75%. The efficiency of Amodiaquine as a standard drug was reported to give 92.5. PMID:25462227

  17. Phycobiliprotein fusion proteins: versatile intensely fluorescent constructs

    NASA Astrophysics Data System (ADS)

    Glazer, Alexander N.; Cai, Yuping A.; Tooley, Aaron J.

    2004-06-01

    Since 1982, phycobiliproteins have served as fluorescent labels in a wide variety of cell and molecule analyses. The exceptional spectroscopic properties of these labels include very high absorbance coefficients and quantum yields, and large Stokes shifts. The spectroscopic diversity of these reagents is restricted to a subset of naturally occurring phycobiliproteins with stable assembly states in vitro, whose target specificity is generated by chemical conjugation to proteins or small molecules. The latter step generates heterogeneity. These limitations have been overcome by expressing various recombinant phycobiliprotein constructs in the cyanobacterium Anabaena sp. PCC7120. Modular recombinant phycobiliprotein-based labels were constructed with some or all of the following features (a) an affinity purification tag; (b) a stable oligomerization domain (to maintain stable higher order assemblies of the phycobiliprotein monomers at very low protein concentration); (c) a biospecific recognition domain. Such phycobiliprotein constructs are readily purified from crude cell extracts by affinity chromatography and used directly as fluorescent labels. To generate constructs for intracellular in vivo labeling, the entire pathways for the biosynthesis of the His-tagged holo- α (phycocyanobilin-bearing) subunit of phycocyanin (emission max. 641 nm) and of the His-tagged holo-α (phycobiliviolin-bearing) subunit of phycoerythrocyanin (emission max. 582 nm) were reconstituted in Escherichia coli.

  18. On the structure of self-affine convex bodies

    SciTech Connect

    Voynov, A S

    2013-08-31

    We study the structure of convex bodies in R{sup d} that can be represented as a union of their affine images with no common interior points. Such bodies are called self-affine. Vallet's conjecture on the structure of self-affine bodies was proved for d = 2 by Richter in 2011. In the present paper we disprove the conjecture for all d≥3 and derive a detailed description of self-affine bodies in R{sup 3}. Also we consider the relation between properties of self-affine bodies and functional equations with a contraction of an argument. Bibliography: 10 titles.

  19. Metal-affinity separations: A new dimension in protein processing

    SciTech Connect

    Arnold, F.H. )

    1991-02-01

    Rapid growth in the preparative and high-resolution analytical applications of metal-affinity chromatography demonstrate the appeal of metal recognition as a basis for protein separations. Stable, inexpensive chelated metals effectively mimic biospecific interactions, providing selective ligands for protein binding. This article reviews recent progress in understanding the mechanisms of metal-protein recognition that underlie metal-affinity separations. Also discussed are schemes for integrating metal-affinity purifications into the expression and bioprocessing of recombinant proteins. Promising future developments include new metal-affinity processes for analytical and preparative-scale separations and a range of techniques for enhancing the selectivity of metal-affinity separations.

  20. Avoiding degenerate coframes in an affine gauge approach to quantum gravity

    SciTech Connect

    Mielke, E.W.; McCrea, J.D.; Ne`eman, Y.; Hehl, F.W.

    1993-04-01

    This report discusses the following concepts on quantum gravity: The affine gauge approach; affine gauge transformations versus active differomorphisms; affine gauge approach to quantum gravity with topology change.

  1. Aluminum monocation basicity and affinity scales.

    PubMed

    Gal, Jean-François; Yáñez, Manuel; Mó, Otilia

    2015-01-01

    The experimental aspects of the determination of thermochemical data for the attachment of the aluminum monocation Al(+) to neutral atoms and molecules are reviewed. Literature aluminum cation affinities (enthalpy scale) and basicities (Gibbs energy scale) are tabulated and discussed. Ab initio quantum chemical calculations at the G4 level on 43 adducts provide a consistent picture of the energetics of the adducts and their structures. The Al(+)-ligand bonding is analyzed in terms of natural bond orbital and atom-in molecule analyses. A brief comparison of the Al(+) basicity scales and other gas- phase cation basicities is presented. PMID:26307732

  2. Contractions of affine Kac-Moody algebras

    NASA Astrophysics Data System (ADS)

    Daboul, J.; Daboul, C.; de Montigny, M.

    2008-08-01

    I review our recent work on contractions of affine Kac-Moody algebras (KMA) and present new results. We study generalized contractions of KMA with respect to their twisted and untwisted KM subalgebras. As a concrete example, we discuss contraction of D(1)4 and D(3)4, based on Z3-grading. We also describe examples of 'level-dependent' contractions, which are based on Z-gradings of KMA. Our work generalizes the Inönü-Wigner contraction of P. Majumdar in several directions. We also give an algorithm for constructing Kac-Moody-like algebras hat g for any Lie algebra g.

  3. Aptamer Affinity Maturation by Resampling and Microarray Selection.

    PubMed

    Kinghorn, Andrew B; Dirkzwager, Roderick M; Liang, Shaolin; Cheung, Yee-Wai; Fraser, Lewis A; Shiu, Simon Chi-Chin; Tang, Marco S L; Tanner, Julian A

    2016-07-19

    Aptamers have significant potential as affinity reagents, but better approaches are critically needed to discover higher affinity nucleic acids to widen the scope for their diagnostic, therapeutic, and proteomic application. Here, we report aptamer affinity maturation, a novel aptamer enhancement technique, which combines bioinformatic resampling of aptamer sequence data and microarray selection to navigate the combinatorial chemistry binding landscape. Aptamer affinity maturation is shown to improve aptamer affinity by an order of magnitude in a single round. The novel aptamers exhibited significant adaptation, the complexity of which precludes discovery by other microarray based methods. Honing aptamer sequences using aptamer affinity maturation could help optimize a next generation of nucleic acid affinity reagents. PMID:27346322

  4. Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

    PubMed Central

    Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

    2012-01-01

    The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

  5. High-affinity Cyclic Peptide Matriptase Inhibitors*

    PubMed Central

    Quimbar, Pedro; Malik, Uru; Sommerhoff, Christian P.; Kaas, Quentin; Chan, Lai Y.; Huang, Yen-Hua; Grundhuber, Maresa; Dunse, Kerry; Craik, David J.; Anderson, Marilyn A.; Daly, Norelle L.

    2013-01-01

    The type II transmembrane serine protease matriptase is a key activator of multiple signaling pathways associated with cell proliferation and modification of the extracellular matrix. Deregulated matriptase activity correlates with a number of diseases, including cancer and hence highly selective matriptase inhibitors may have therapeutic potential. The plant-derived cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), is a promising drug scaffold with potent matriptase inhibitory activity. In the current study we have analyzed the structure-activity relationships of SFTI-1 and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a structurally divergent trypsin inhibitor from Momordica cochinchinensis that also contains a cyclic backbone. We show that MCoTI-II is a significantly more potent matriptase inhibitor than SFTI-1 and that all alanine mutants of both peptides, generated using positional scanning mutagenesis, have decreased trypsin affinity, whereas several mutations either maintain or result in enhanced matriptase inhibitory activity. These intriguing results were used to design one of the most potent matriptase inhibitors known to date with a 290 pm equilibrium dissociation constant, and provide the first indication on how to modulate affinity for matriptase over trypsin in cyclic peptides. This information might be useful for the design of more selective and therapeutically relevant inhibitors of matriptase. PMID:23548907

  6. Heparin affinity purification of extracellular vesicles

    PubMed Central

    Balaj, Leonora; Atai, Nadia A.; Chen, Weilin; Mu, Dakai; Tannous, Bakhos A.; Breakefield, Xandra O.; Skog, Johan; Maguire, Casey A.

    2015-01-01

    Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity. PMID:25988257

  7. Affine conformal vectors in space-time

    NASA Astrophysics Data System (ADS)

    Coley, A. A.; Tupper, B. O. J.

    1992-05-01

    All space-times admitting a proper affine conformal vector (ACV) are found. By using a theorem of Hall and da Costa, it is shown that such space-times either (i) admit a covariantly constant vector (timelike, spacelike, or null) and the ACV is the sum of a proper affine vector and a conformal Killing vector or (ii) the space-time is 2+2 decomposable, in which case it is shown that no ACV can exist (unless the space-time decomposes further). Furthermore, it is proved that all space-times admitting an ACV and a null covariantly constant vector (which are necessarily generalized pp-wave space-times) must have Ricci tensor of Segré type {2,(1,1)}. It follows that, among space-times admitting proper ACV, the Einstein static universe is the only perfect fluid space-time, there are no non-null Einstein-Maxwell space-times, and only the pp-wave space-times are representative of null Einstein-Maxwell solutions. Otherwise, the space-times can represent anisotropic fluids and viscous heat-conducting fluids, but only with restricted equations of state in each case.

  8. Fatigue damage prognosis using affine arithmetic

    NASA Astrophysics Data System (ADS)

    Gbaguidi, Audrey; Kim, Daewon

    2014-02-01

    Among the essential steps to be taken in structural health monitoring systems, damage prognosis would be the field that is least investigated due to the complexity of the uncertainties. This paper presents the possibility of using Affine Arithmetic for uncertainty propagation of crack damage in damage prognosis. The structures examined are thin rectangular plates made of titanium alloys with central mode I cracks and a composite plate with an internal delamination caused by mixed mode I and II fracture modes, under a harmonic uniaxial loading condition. The model-based method for crack growth rates are considered using the Paris Erdogan law model for the isotropic plates and the delamination growth law model proposed by Kardomateas for the composite plate. The parameters for both models are randomly taken and their uncertainties are considered as defined by an interval instead of a probability distribution. A Monte Carlo method is also applied to check whether Affine Arithmetic (AA) leads to tight bounds on the lifetime of the structure.

  9. Exploring Fluorous Affinity by Liquid Chromatography.

    PubMed

    Catani, Martina; Guzzinati, Roberta; Marchetti, Nicola; Pasti, Luisa; Cavazzini, Alberto

    2015-07-01

    Terms such as "fluorous affinity" and "fluorophilicity" have been used to describe the unique partition and sorption properties often exhibited by highly fluorinated organic compounds, that is molecules rich in sp(3) carbon-fluorine bonds. In this work, we made use of a highly fluorinated stationary phase and a series of benzene derivatives to study the effect of one single perfluorinated carbon on the chromatographic behavior and adsorption properties of molecules. For this purpose, the adsorption equilibria of α,α,α-trifluorotoluene, toluene, and other alkylbenzenes have been studied by means of nonlinear chromatography in a variety of acetonitrile/water eluents. Our results reveal that one single perfluorinated carbon is already enough to induce a drastic change in the adsorption properties of molecules on the perfluorinated stationary phase. In particular, it has been found that adsorption is monolayer if the perfluoroalkyl carbon is present but that, when this unit is missing, molecules arrange as multilayer stack structures. These findings can contribute to the understanding of molecular mechanisms of fluorous affinity. PMID:26047527

  10. Quantification of hydrophobic interaction affinity of colloids

    NASA Astrophysics Data System (ADS)

    Saini, G.; Nasholm, N.; Wood, B. D.

    2009-12-01

    Colloids play an important role in a wide variety of disciplines, including water and wastewater treatment, subsurface transport of metals and organic contaminants, migration of fines in oil reservoirs, biocolloid (virus and bacteria) transport in subsurface, and are integral to laboratory transport studies. Although the role of hydrophobicity in adhesion and transport of colloids, particularly bacteria, is well known; there is scarcity of literature regarding hydrophobicity measurement of non-bacterial colloids and other micron-sized particles. Here we detail an experimental approach based on differential partitioning of colloids between two liquid phases (hydrocarbon and buffer) as a measure of the hydrophobic interaction affinity of colloids. This assay, known as Microbial adhesion to hydrocarbons or MATH, is frequently used in microbiology and bacteriology for quantifying the hydrophobicity of microbes. Monodispersed colloids and particles, with sizes ranging from 1 micron to 33 micron, were used for the experiments. A range of hydrophobicity values were observed for different particles. The hydrophobicity results are also verified against water contact angle measurements of these particles. This liquid-liquid partitioning assay is quick, easy-to-perform and requires minimal instrumentation. Estimation of the hydrophobic interaction affinity of colloids would lead to a better understanding of their adhesion to different surfaces and subsequent transport in porous media.

  11. Using Fluorescence Spectroscopy to Evaluate Hill Parameters and Heterogeneity of Ligand Binding to Cytochromes P450

    NASA Astrophysics Data System (ADS)

    Marsch, Glenn A.; Carlson, Benjamin; Hansen, Jennifer; Mihelc, Elaine; Martin, Martha V.; Guengerich, F. Peter

    2009-03-01

    The cytochromes P450 (CYPs) are hemoproteins that oxidize many drugs and carcinogens. Binding interactions of two CYPs with Nile Red, pyrene, and alpha-naphthoflavone were studied using fluorescence quenching. Upon interaction with CYPs, fluorescence from pyrene excited-state dimers was quenched more efficiently than fluorescence from pyrene monomers. Quenching data was fit to the Hill equation to determine binding affinities and the Hill parameter n for the interaction of substrates with CYPs. All ligands showed strong binding to the CYPs, especially alpha-naphthoflavone, but exhibited little or no cooperativity in the interaction. Modified Stern-Volmer plots were used to confirm binding affinities, and suggested heterogeneous populations of amino acid fluorophores. Fluorescence anisotropy experiments suggest that CYP molecules tumble more rapidly when alpha-naphthoflavone is added.

  12. Application of silica fume as a new SP-extractor for trace determination of Zn(II) and Cd(II) in pharmaceutical and environmental samples by square-wave anodic stripping voltammetry

    NASA Astrophysics Data System (ADS)

    Ahmed, Salwa A.; Gaber, Ahmed A. Abdel; Rahim, Asmaa M. Abdel

    2015-04-01

    In this work, silica fume (SF) is used as a solid-phase extractor for extraction of Zn(II) and Cd(II) from aqueous solutions. Characterization of SF is performed by Fourier transform infrared, X-ray diffraction, transmission and scanning electron microscopy. The optimum experimental conditions for the two metal ions are investigated using batch and column techniques. The maximum adsorption capacity values are found to be 54.13 and 121.28 mg g-1 at the optimum pH 6.0 and 8.0 for Zn(II) and Cd(II), respectively. The equilibrium data are analyzed using the Langmuir, Freundlich, and Temkin isotherms by nonlinear regression analysis. Also, the kinetics analysis revealed that the overall adsorption process is successfully fitted with the pseudo-second-order model. The method is applied for determination of the target metal ions in pharmaceutical and environmental samples using square-wave anodic stripping voltammetry. The limit of detection (LOD) values are 0.102 and 1.43 × 10-3 mg L-1 for Zn(II) and Cd(II), respectively. The percentage recovery values are 98.8-100.5 % which indicate the success of the proposed method for determination of Zn(II) and Cd(II) without interfering effects.

  13. Specificity and Ligand Affinities of the Cocaine Aptamer: Impact of Structural Features and Physiological NaCl.

    PubMed

    Sachan, Ashish; Ilgu, Muslum; Kempema, Aaron; Kraus, George A; Nilsen-Hamilton, Marit

    2016-08-01

    The cocaine aptamer has been seen as a good candidate for development as a probe for cocaine in many contexts. Here, we demonstrate that the aptamer binds cocaine, norcocaine, and cocaethylene with similar affinities and aminoglycosides with similar or higher affinities in a mutually exclusive manner with cocaine. Analysis of its affinities for a series of cocaine derivatives shows that the aptamer specificity is the consequence of its interaction with all faces of the cocaine molecule. Circular dichroism spectroscopy and 2-aminopurine (2AP) fluorescence studies show no evidence of large structural rearrangement of the cocaine aptamer upon ligand binding, which is contrary to the general view of this aptamer. The aptamer's affinity for cocaine and neomycin-B decreases with the inclusion of physiological NaCl. The substitution of 2AP for A in position 6 (2AP6) of the aptamer sequence eliminated the effect of NaCl on its affinities for cocaine and analogues, but not for neomycin-B, showing a selective effect of 2AP substitution on cocaine binding. The affinity for cocaine also decreased with increasing concentrations of serum or urine, with the 2AP6 substitution blunting the effect of urine. Its low affinities for cocaine and metabolites and its ability to bind irrelevant compounds limit the opportunities for application of this aptamer in its current form as a selective and reliable sensor for cocaine. However, these studies also show that a small structural adjustment to the aptamer (2AP exchanged for adenine) can increase its specificity for cocaine in physiological NaCl relative to an off-target ligand. PMID:27348073

  14. Characterization of a dockerin-based affinity tag: application for purification of a broad variety of target proteins.

    PubMed

    Demishtein, Alik; Karpol, Alon; Barak, Yoav; Lamed, Raphael; Bayer, Edward A

    2010-01-01

    Cellulose, a major component of plant matter, is degraded by a cell surface multiprotein complex called the cellulosome produced by several anaerobic bacteria. This complex coordinates the assembly of different glycoside hydrolases, via a high-affinity Ca(2+)-dependent interaction between the enzyme-borne dockerin and the scaffoldin-borne cohesin modules. In this study, we characterized a new protein affinity tag, ΔDoc, a truncated version (48 residues) of the Clostridium thermocellum Cel48S dockerin. The truncated dockerin tag has a binding affinity (K(A)) of 7.7 × 10(8)M(-1), calculated by a competitive enzyme-linked assay system. In order to examine whether the tag can be used for general application in affinity chromatography, it was fused to a range of target proteins, including Aequorea victoria green fluorescent protein (GFP), C. thermocellum β-glucosidase, Escherichia coli thioesterase/protease I (TEP1), and the antibody-binding ZZ-domain from Staphylococcus aureus protein A. The results of this study significantly extend initial studies performed using the Geobacillus stearothermophilus xylanase T-6 as a model system. In addition, the enzymatic activity of a C. thermocellum β-glucosidase, purified using this approach, was tested and found to be similar to that of a β-glucosidase preparation (without the ΔDoc tag) purified using the standard His-tag. The truncated dockerin derivative functioned as an effective affinity tag through specific interaction with a cognate cohesin, and highly purified target proteins were obtained in a single step directly from crude cell extracts. The relatively inexpensive beaded cellulose-based affinity column was reusable and maintained high capacity after each cycle. This study demonstrates that deletion into the first Ca(2+)-binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification. PMID:21038354

  15. Fluorescence Live Cell Imaging

    PubMed Central

    Ettinger, Andreas

    2014-01-01

    Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio, and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities, and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate fluorescent protein constructs by spinning disk confocal microscopy. PMID:24974023

  16. Fluorescent fiber diagnostics

    DOEpatents

    Toeppen, John S.

    1994-10-04

    A fluorescent fiber (13) having a doped core (16) is pumped (11) by light (18) of a relatively short wavelength to produce fluorescence at a longer wavelength that is detected by detector (24). The level of fluorescence is monitored (26) and evaluated to provide information as to the excitation of the fiber (13) or the environment thereof. In particular, the level of intensity of the detected fluorescence may be used to measure the intensity of a light beam (18) passing axially through an optical fiber system (12) (FIG. 1 ), or the intensity of a light beam (46) passing radially through a fluorescent fiber (13) (FIG. 2 ), or the level of a fluid (32) in a tank (31) (FIG. 3 ), or a scintillation event (37) in a fluorescent fiber (13) pumped to produce amplification of the scintillation event (FIG. 4 ).

  17. Fluorescent fiber diagnostics

    DOEpatents

    Toeppen, John S.

    1994-01-01

    A fluorescent fiber (13) having a doped core (16) is pumped (11) by light (18) of a relatively short wavelength to produce fluorescence at a longer wavelength that is detected by detector (24). The level of fluorescence is monitored (26) and evaluated to provide information as to the excitation of the fiber (13) or the environment thereof. In particular, the level of intensity of the detected fluorescence may be used to measure the intensity of a light beam (18) passing axially through an optical fiber system (12) (FIG. 1 ), or the intensity of a light beam (46) passing radially through a fluorescent fiber (13) (FIG. 2 ), or the level of a fluid (32) in a tank (31) (FIG. 3 ), or a scintillation event (37) in a fluorescent fiber (13) pumped to produce amplification of the scintillation event (FIG. 4 ).

  18. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach. PMID:27498895

  19. Novel fluorescent protein from Hydnophora rigida possesses green emission.

    PubMed

    Idrees, M; Thangavelu, K; Sikaroodi, M; Smith, C; Sivaraman, J; Gillevet, P M; Bokhari, H

    2014-05-23

    Fluorescent proteins are a family of proteins capable of producing fluorescence at various specific wavelengths of ultra violet light. We have previously reported the identification and characterization of a novel cyan fluorescent protein (HriCFP) from a reef coral species, Hydnophora rigida. In search of new members of the diverse family of fluorescent proteins, here we report a new green fluorescent protein (HriGFP) from H. rigida. HriGFP was identified, cloned, expressed in Escherichia coli and purified to homogeneity by metal affinity and size exclusion chromatography. The dynamic light scattering and gel filtration experiments suggested the presence of monomers in solution. The peptide mass fingerprint on the purified protein established the identity of HriGFP. HriGFP had excitation peak at 507 nm and emission peak at 527 nm. HriGFP was similar to HriCFP except the last 16 amino acid sequence at the C-terminal; however, they have shown least similarity with other known fluorescent proteins. Moreover the computational model suggests that HriGFP is a globular protein which consists of 6 α-helices and 3 β-sheets. Taken together our results suggested that HriGFP is a novel naturally occurring fluorescent protein that exists as a monomer in solution. PMID:24747076

  20. Bare magnetic nanoparticles as fluorescence quenchers for detection of thrombin.

    PubMed

    Yu, Jiemiao; Yang, Liangrong; Liang, Xiangfeng; Dong, Tingting; Liu, Huizhou

    2015-06-21

    Rapid and sensitive detection of thrombin has very important significance in clinical diagnosis. In this work, bare magnetic iron oxide nanoparticles (magnetic nanoparticles) without any modification were used as fluorescence quenchers. In the absence of thrombin, a fluorescent dye (CY3) labeled thrombin aptamer (named CY3-aptamer) was adsorbed on the surface of magnetic nanoparticles through interaction between a phosphate backbone of the CY3-aptamer and hydroxyl groups on the bare magnetic nanoparticles in binding solution, leading to fluorescence quenching. Once thrombin was introduced, the CY3-aptamer formed a G-quartet structure and combined with thrombin, which resulted in the CY3-aptamer being separated from the magnetic nanoparticles and restoration of fluorescence. This proposed assay took advantage of binding affinity between the CY3-aptamer and thrombin for specificity, and bare magnetic nanoparticles for fluorescence quenching. The fluorescence signal had a good linear relationship with thrombin concentration in the range of 1-60 nM, and the limit of detection for thrombin was estimated as low as 0.5 nM. Furthermore, this method could be applied for other target detection using the corresponding fluorescence labeled aptamer. PMID:25894923

  1. Botany: floral fluorescence effect.

    PubMed

    Gandía-Herrero, Fernando; García-Carmona, Francisco; Escribano, Josefa

    2005-09-15

    The way flowers appear to insects is crucial for pollination. Here we describe an internal light-filtering effect in the flowers of Mirabilis jalapa, in which the visible fluorescence emitted by one pigment, a yellow betaxanthin, is absorbed by another, a violet betacyanin, to create a contrasting fluorescent pattern on the flower's petals. This finding opens up new possibilities for pollinator perception as fluorescence has not previously been considered as a potential signal in flowers. PMID:16163341

  2. Fluorescent minerals, a review

    USGS Publications Warehouse

    Modreski, P.J.; Aumente-Modreski, R.

    1996-01-01

    Fluorescent minerals are more than just an attractive novelty, and collecting them is a speciality for thousands of individuals who appreciate their beauty, rarity, and scientific value. Fluorescent properties can be used as an aid to mineral identification, locality determination, and distinction between natural and synthetic gemstones. This article gives an overview of those aspects of fluorescence that are of most interest to collectors, hobbyists, and mineralogists. -from Authors

  3. Fluorescence lifetime imaging of coral fluorescent proteins.

    PubMed

    Cox, Guy; Matz, Mikhail; Salih, Anya

    2007-03-01

    Corals, like many other coelenterates, contain fluorescent pigments that show considerable homology with the well known green fluorescent protein of the jellyfish Aequoria. In corals, unlike jellyfish, multiple proteins are present and the range of excitations and emissions suggest the possibility of energy transfer. The occurrence of Förster resonant energy transfer (FRET) between fluorescent proteins in corals has already been reported and time-resolved spectra have shown the effect on fluorescent lifetime, but without any spatial resolution. Lifetime confocal microscopy offers lower time resolution but excellent spatial resolution. Lifetimes of the isolated A. millepora pigments amilFP490, amilFP504, and amilFP593 (names indicate emission peaks) were 2.8, 2.9, and 2.9 ns, respectively. In the coral sample, imaging the entire emission spectrum from 420 nm, the mean lifetime was reduced to 1.5 ns, implying that FRET was occurring. Looking just at the fluorescence from FRET donors the lifetime was even shorter, at 1.3 ns, supporting this interpretation. In contrast, no reduction in lifetime is seen in the coral Euphyllia ancora, where the pigment distribution also suggests that the pigments are unlikely to be involved in photoprotection. This study set out to determine the extent of FRET between pigments in two corals, Acropora millepora and Euphyllia, ancora which differ in the arrangement of their pigments and hence possibly in pigment function. PMID:17279514

  4. Factors that influence T box riboswitch efficacy and tRNA affinity.

    PubMed

    Zeng, C; Zhou, S; Bergmeier, S C; Hines, J V

    2015-09-01

    The T box riboswitch is an intriguing potential target for antibacterial drug discovery. Found primarily in Gram-positive bacteria, the riboswitch regulates gene expression by selectively responding to uncharged tRNA to control transcription readthrough. Polyamines and molecular crowding are known to specifically affect RNA function, but their effect on T box riboswitch efficacy and tRNA affinity have not been fully characterized. A fluorescence-monitored in vitro transcription assay was developed to readily quantify these molecular interactions and to provide a moderate-throughput functional assay for a comprehensive drug discovery screening cascade. The polyamine spermidine specifically enhanced T box riboswitch readthrough efficacy with an EC50 = 0.58 mM independent of tRNA binding. Molecular crowding, simulated by the addition of polyethylene glycol, had no effect on tRNA affinity for the riboswitch, but did reduce the efficacy of tRNA-induced readthrough. These results indicate that the T box riboswitch tRNA affinity and readthrough efficacy are intricately modulated by environmental factors. PMID:26220520

  5. Biotinylated Cyclophane: Synthesis, Cyclophane-Avidin Conjugates, and Their Enhanced Guest-Binding Affinity.

    PubMed

    Hayashida, Osamu; Kojima, Miwa; Kusano, Shuhei

    2015-10-01

    Cationic and anionic cyclophanes bearing a biotin moiety were synthesized as a water-soluble host (1a and 1b, respectively). Both hosts 1a and 1b were found to strongly bind avidin with binding constants of 1.3 × 10(8) M(-1), as confirmed by surface plasmon resonance measurements. The present conjugate of 1a with avidin (1a-avidin) showed an enhanced guest binding affinity toward fluorescence guests such as TNS and 2,6-ANS. The K values of 1a-avidin conjugate with TNS and 2,6-ANS were ~19-fold larger than those of monocyclic cyclophane 1a with the identical guests. Favorable hydrophobic and electrostatic interactions between 1a-avidin and TNS were suggested by computer-aided molecular modeling calculations. Moreover, addition of excess biotin to the complexes of 1a-avidin with the guests resulted in dissociation of 1a-avidin to avidin and 1a having less guest-binding affinity. Conversely, such enhancements in the guest-binding affinity were not obviously observed for the conjugate of anionic 1b with avidin (1b-avidin) due to electrostatic repulsion between anionic 1b and anionic guests. PMID:26360807

  6. Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers.

    PubMed

    Martin, Jennifer A; Chushak, Yaroslav; Chávez, Jorge L; Hagen, Joshua A; Kelley-Loughnane, Nancy

    2016-01-01

    Immobilization of nucleic acid aptamer recognition elements selected free in solution onto the surface of biosensor platforms has proven challenging. This study investigated the binding of multiple aptamer/target pairs immobilized on a commercially available microarray as a model system mimicking biosensor applications. The results indicate a minimum distance (linker length) from the surface and thymine nucleobase linker provides reproducible binding across varying conditions. An indirect labeling method, where the target was labeled with a biotin followed by a brief Cy3-streptavidin incubation, provided a higher signal-to-noise ratio and over two orders of magnitude improvement in limit of detection, compared to direct Cy3-protein labeling. We also showed that the affinities of the aptamer/target interaction can change between direct and indirect labeling and conditions to optimize for the highest fluorescence intensity will increase the sensitivity of the assay but will not change the overall affinity. Additionally, some sequences which did not initially bind demonstrated binding when conditions were optimized. These results, in combination with studies demonstrating enhanced binding in nonselection buffers, provided insights into the structure and affinity of aptamers critical for biosensor applications and allowed for generalizations in starting conditions for researchers wishing to investigate aptamers on a microarray surface. PMID:27042344

  7. Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers

    PubMed Central

    Martin, Jennifer A.; Chushak, Yaroslav; Chávez, Jorge L.; Hagen, Joshua A.; Kelley-Loughnane, Nancy

    2016-01-01

    Immobilization of nucleic acid aptamer recognition elements selected free in solution onto the surface of biosensor platforms has proven challenging. This study investigated the binding of multiple aptamer/target pairs immobilized on a commercially available microarray as a model system mimicking biosensor applications. The results indicate a minimum distance (linker length) from the surface and thymine nucleobase linker provides reproducible binding across varying conditions. An indirect labeling method, where the target was labeled with a biotin followed by a brief Cy3-streptavidin incubation, provided a higher signal-to-noise ratio and over two orders of magnitude improvement in limit of detection, compared to direct Cy3-protein labeling. We also showed that the affinities of the aptamer/target interaction can change between direct and indirect labeling and conditions to optimize for the highest fluorescence intensity will increase the sensitivity of the assay but will not change the overall affinity. Additionally, some sequences which did not initially bind demonstrated binding when conditions were optimized. These results, in combination with studies demonstrating enhanced binding in nonselection buffers, provided insights into the structure and affinity of aptamers critical for biosensor applications and allowed for generalizations in starting conditions for researchers wishing to investigate aptamers on a microarray surface. PMID:27042344

  8. Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose.

    PubMed

    Jia, Yinshan; Jarrett, Harry W

    2015-08-01

    The use of a method of coupling DNA was investigated for trapping and purifying transcription factors. Using the GFP-C/EBP (CAAT/enhancer binding protein) fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry used is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA binding. The method involves introducing a ribose nucleotide to the 3' end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose that couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture, and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes, including E2A, c-Myc, and Myo-D, were also purified, but myogenin and NFκB were not. Therefore, this approach proved to be valuable for both affinity chromatography and the trapping approach. PMID:25935261

  9. Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose

    PubMed Central

    Jia, Yinshan; Jarrett, Harry W.

    2015-01-01

    The uses of a method of coupling DNA is investigated for trapping and purifying transcription factors. Using the GFP-C/EBP fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry utilized is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA-binding. The method involves introducing a ribose nucleotide to the 3′ end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose which couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes including E2A, c-myc, and myo-D were also purified but myogenenin and NFκB were not. Therfore, this approach proved valuable for both affinity chromatography and for the trapping approach. PMID:25935261

  10. SnAvi--a new tandem tag for high-affinity protein-complex purification.

    PubMed

    Schäffer, Ursula; Schlosser, Andreas; Müller, Kristian M; Schäfer, Angelika; Katava, Nenad; Baumeister, Ralf; Schulze, Ekkehard

    2010-04-01

    Systematic tandem-affinity-purification (TAP) of protein complexes was tremendously successful in yeast and has changed the general concept of how we understand protein function in eukaryotic cells. The transfer of this method to other model organisms has been difficult and may require specific adaptations. We were especially interested to establish a cell-type-specific TAP system for Caenorhabditis elegans, a model animal well suited to high-throughput analysis, proteomics and systems biology. By combining the high-affinity interaction between in vivo biotinylated target-proteins and streptavidin with the usage of a newly identified epitope of the publicly shared SB1 monoclonal antibody we created a novel in vivo fluorescent tag, the SnAvi-Tag. We show the versatile application of the SnAvi-Tag in Escherichia coli, vertebrate cells and in C. elegans for tandem affinity purification of protein complexes, western blotting and also for the in vivo sub-cellular localization of labelled proteins. PMID:20047968

  11. Dual-emission fluorescent sensor based on AIE organic nanoparticles and Au nanoclusters for the detection of mercury and melamine

    NASA Astrophysics Data System (ADS)

    Niu, Caixia; Liu, Qiuling; Shang, Zhehai; Zhao, Liu; Ouyang, Jin

    2015-04-01

    A novel dual-emission ratiometric fluorescence probe is designed and developed by linking two parts, positively charged aggregation-induced emission (AIE) organic fluorescence nanoparticles (OFNs) as the reference and negatively charged Au nanoclusters (Au NCs) as the response, by electrostatic attraction for the first time. This probe can be used for not only visual but quantitative determination of Hg2+ as well as melamine, because red fluorescence of Au NCs can be quenched by mercury ions and recovered by melamine, due to the strong affinity metallophilic Hg2+-Au interaction and stronger affinity Hg2+-N. During this process, the green fluorescence of AIE-OFNs remains constant owing to the protection of ε-polylysine (ε-Ply). In addition, the prepared dual-emission ratiometric fluorescence probe has good biocompatibility, indicating the potential of the probe in applications of biological imaging and detection. The results revealed that this dual-emission ratiometric fluorescence probe broadens the application of AIE-based organic fluorescent nanoparticles, and presents a new method to prepare more sensitive, biocompatible, and visual ratiometric fluorescent probes.A novel dual-emission ratiometric fluorescence probe is designed and developed by linking two parts, positively charged aggregation-induced emission (AIE) organic fluorescence nanoparticles (OFNs) as the reference and negatively charged Au nanoclusters (Au NCs) as the response, by electrostatic attraction for the first time. This probe can be used for not only visual but quantitative determination of Hg2+ as well as melamine, because red fluorescence of Au NCs can be quenched by mercury ions and recovered by melamine, due to the strong affinity metallophilic Hg2+-Au interaction and stronger affinity Hg2+-N. During this process, the green fluorescence of AIE-OFNs remains constant owing to the protection of ε-polylysine (ε-Ply). In addition, the prepared dual-emission ratiometric fluorescence probe

  12. Structure of a High-Affinity

    SciTech Connect

    Saphire, E.O.; Montero, M.; Menendez, A.; Houten, N.E.van; Irving, M.B.; Pantophlet, R.; Swick, M.B.; Parren, P.W.H.I.; Burton, D.R.; Scott, J.K.; Wilson, I.A.; /Scripps Res. Inst. /Simon Fraser U. /British Columbia U.

    2007-07-13

    The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 Angstrom resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining

  13. hPEPT1 affinity and translocation of selected Gln-Sar and Glu-Sar dipeptide derivatives.

    PubMed

    Eriksson, André Huss; Elm, Peter L; Begtrup, Mikael; Nielsen, Robert; Steffansen, Bente; Brodin, Birger

    2005-01-01

    The intestinal di- and tripeptide transporter hPEPT1 is considered responsible for the absorption of di- and tripeptides arising from digestion, along with several drugs and prodrugs. In order to gather information on the binding site of the protein, several structure-affinity relationships have been suggested. However, these are not necessarily predictive of compounds that are actually translocated by hPEPT1. More information on affinity to and translocation via hPEPT1 of side-chain-modified dipeptides may be gained by conducting a study of selected dipeptide derivatives with variety in size, hydrophobicity, and bond type. The aim of the present study was to synthesize new esters and amides based on L-Glu-Sar and investigate the effects that bond type and size of modification of the N-terminal side chain of sarcosine-containing dipeptides have on the affinity to and translocation via hPEPT1. The esters L-Glu(O-i-Bu)-Sar and L-Glu(OCH(2)Ada)-Sar and the amides L-Gln(N,N-dimethyl)-Sar and L-Gln(N-piperidinyl)-Sar were synthesized, and affinity to and translocation via hPEPT1 were investigated in mature Caco-2 cell monolayers, grown on permeable supports. Affinity was estimated in a competition assay using (14)C-labeled Gly-Sar. Translocation was measured as fluorescence ratios induced by the substrates using the fluorescent probe BCECF and an epifluorescence microscope setup. All compounds showed high affinity to hPEPT1, but only the amides L-Gln(N,N-dimethyl)-Sar and L-Gln(N-piperidinyl)-Sar were translocated by hPEPT1. hPEPT1 is very susceptible to modifications of the N-terminal amino acid side chain of dipeptidomimetic substrates, in terms of achieving compounds with high affinity for the transporter. However, as affinity is not predictive of translocation, derivatization in this position must be performed with great caution since some of the compounds investigated turn out not to be translocated by the transporter. PMID:15934785

  14. Automatic gesture analysis using constant affine velocity.

    PubMed

    Cifuentes, Jenny; Boulanger, Pierre; Pham, Minh Tu; Moreau, Richard; Prieto, Flavio

    2014-01-01

    Hand human gesture recognition has been an important research topic widely studied around the world, as this field offers the ability to identify, recognize, and analyze human gestures in order to control devices or to interact with computer interfaces. In particular, in medical training, this approach is an important tool that can be used to obtain an objective evaluation of a procedure performance. In this paper, some obstetrical gestures, acquired by a forceps, were studied with the hypothesis that, as the scribbling and drawing movements, they obey the one-sixth power law, an empirical relationship which connects path curvature, torsion, and euclidean velocity. Our results show that obstetrical gestures have a constant affine velocity, which is different for each type of gesture and based on this idea this quantity is proposed as an appropriate classification feature in the hand human gesture recognition field. PMID:25570332

  15. Effectively nonlocal metric-affine gravity

    NASA Astrophysics Data System (ADS)

    Golovnev, Alexey; Koivisto, Tomi; Sandstad, Marit

    2016-03-01

    In metric-affine theories of gravity such as the C-theories, the spacetime connection is associated to a metric that is nontrivially related to the physical metric. In this article, such theories are rewritten in terms of a single metric, and it is shown that they can be recast as effectively nonlocal gravity. With some assumptions, known ghost-free theories with nonsingular and cosmologically interesting properties may be recovered. Relations between different formulations are analyzed at both perturbative and nonperturbative levels, taking carefully into account subtleties with boundary conditions in the presence of integral operators in the action, and equivalences between theories related by nonlocal redefinitions of the fields are verified at the level of equations of motion. This suggests a possible geometrical interpretation of nonlocal gravity as an emergent property of non-Riemannian spacetime structure.

  16. Affinities of the Swartkrans early Homo mandibles.

    PubMed

    Curnoe, Darren

    2008-01-01

    The southern African early Homo assemblage continues to make important contributions to understanding the systematics, adaptations and evolutionary history of the human genus. However, the taxonomy of this sample is in a state of flux. This study examines the size and shape of the mandibular bodies of Swartkrans SK 15 and SK 45 comparing them with variation in two early Homo taxa (H. habilis sensu lato and H. sapiens erectus). The research aims to clarify their phenetic affinities and systematics through univariate statistics, inferential testing and multivariate analysis employing size (Log-transformed) and shape (Mosimann variables). Neither of them strongly resembles H. habilis sensu lato or H. sapiens erectus, rather, they probably sample a novel species of Homo not seen in East Africa. Moreover, there is considerable morphological variability within the Swartkrans sample and the possibility of more than one novel species being sampled at this site cannot be excluded. PMID:18402959

  17. Wetting on rough self-affine surfaces

    NASA Astrophysics Data System (ADS)

    Palasantzas, George

    1995-05-01

    In this paper, we present a general investigation of the effective potential for complete wetting on self-affine rough surfaces. The roughness effect is investigated by means of the height-height correlation model in Fourier space ~(1+aξ2q2)-1-H. The parameters H and ξ are, respectively, the roughness exponent and the substrate in-plane correlation length. It is observed that the effect of H on the free interface profile is significant for ξ>ξ) regime is characterized by a power-law scaling ~Y-2.

  18. Dye affinity cryogels for plasmid DNA purification.

    PubMed

    Çimen, Duygu; Yılmaz, Fatma; Perçin, Işık; Türkmen, Deniz; Denizli, Adil

    2015-11-01

    The aim of this study is to prepare megaporous dye-affinity cryogel discs for the purification of plasmid DNA (pDNA) from bacterial lysate. Poly(hydroxyethyl methacrylate) [PHEMA] cryogel discs were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) redox pair in an ice bath. Cibacron Blue F3GA was used as an affinity ligand (loading amount: 68.9μmol/g polymer). The amount of pDNA adsorbed onto the PHEMA-Cibacron Blue F3GA cryogel discs first increased and then reached a plateau value (i.e., 32.5mg/g cryogel) at 3.0mg/mL pDNA concentration. Compared with the PHEMA cryogel (0.11mg/g cryogel), the pDNA adsorption capacity of the PHEMA-Cibacron Blue F3GA cryogel (32.4mg/g polymer) was improved significantly due to the Cibacron Blue 3GA immobilization onto the polymeric matrix. pDNA adsorption amount decreased from 11.7mg/g to 1.1mg/g with the increasing of NaCl concentration. The maximum pDNA adsorption was achieved at 4°C. The overall recovery of pDNA was calculated as 90%. The PHEMA-Cibacron Blue F3GA cryogel discs could be used five times without decreasing the pDNA adsorption capacity significantly. The results show that the PHEMA-Cibacron Blue F3GA cryogel discs promise high selectivity for pDNA. PMID:26249596

  19. In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation

    PubMed Central

    Li, Bing; Fouts, Ashley E; Stengel, Katharina; Luan, Peng; Dillon, Michael; Liang, Wei-Ching; Feierbach, Becket; Kelley, Robert F; Hötzel, Isidro

    2014-01-01

    Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (KD < 10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid

  20. Prediction of Neutral Salt Elution Profiles for Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Stellwagen, Earle

    1981-04-01

    Neutral salts exhibit very marked differences as eluants of proteins from affinity columns. We observe: (i) that the relative potencies of neutral salts as eluants are independent of the protein or the affinity ligand in the systems studied, (ii) that the absolute salt concentration necessary to elute any given protein bound to the affinity matrix is proportional to the algebraic sum of a set of elution coefficients defined herein for the separate ions present in the solution, and (iii) that the proportionality between elution potency and elution coefficient is a function of the affinity of the protein for the immobilized ligand. Given the concentration of one neutral salt required for elution of a protein of interest from an affinity column, the elution capability of any neutral salt at any temperature can be quantitatively predicted for that protein. Accordingly, application and elution protocols for affinity chromatography can be designed to optimize the yield and fold purification of proteins.

  1. Fluorescence in insects

    NASA Astrophysics Data System (ADS)

    Welch, Victoria L.; Van Hooijdonk, Eloise; Intrater, Nurit; Vigneron, Jean-Pol

    2012-10-01

    Fluorescent molecules are much in demand for biosensors, solar cells, LEDs and VCSEL diodes, therefore, considerable efforts have been expended in designing and tailoring fluorescence to specific technical applications. However, naturally occurring fluorescence of diverse types has been reported from a wide array of living organisms: most famously, the jellyfish Aequorea victoria, but also in over 100 species of coral and in the cuticle of scorpions, where it is the rule, rather than the exception. Despite the plethora of known insect species, comparatively few quantitative studies have been made of insect fluorescence. Because of the potential applications of natural fluorescence, studies in this field have relevance to both physics and biology. Therefore, in this paper, we review the literature on insect fluorescence, before documenting its occurrence in the longhorn beetles Sternotomis virescens, Sternotomis variabilis var. semi rufescens, Anoplophora elegans and Stellognatha maculata, the tiger beetles Cicindela maritima and Cicindela germanica and the weevil Pachyrrhynchus gemmatus purpureus. Optical features of insect fluorescence, including emitted wavelength, molecular ageing and naturally occurring combinations of fluorescence with bioluminescence and colour-producing structures are discussed.

  2. Affine Vertex Operator Algebras and Modular Linear Differential Equations

    NASA Astrophysics Data System (ADS)

    Arike, Yusuke; Kaneko, Masanobu; Nagatomo, Kiyokazu; Sakai, Yuichi

    2016-05-01

    In this paper, we list all affine vertex operator algebras of positive integral levels whose dimensions of spaces of characters are at most 5 and show that a basis of the space of characters of each affine vertex operator algebra in the list gives a fundamental system of solutions of a modular linear differential equation. Further, we determine the dimensions of the spaces of characters of affine vertex operator algebras whose numbers of inequivalent simple modules are not exceeding 20.

  3. Affinity purification of recombinant proteins using a novel silica-binding peptide as a fusion tag.

    PubMed

    Abdelhamid, Mohamed A A; Motomura, Kei; Ikeda, Takeshi; Ishida, Takenori; Hirota, Ryuichi; Kuroda, Akio

    2014-06-01

    We recently reported that silica is deposited on the coat of Bacillus cereus spores as a layer of nanometer-sized particles (Hirota et al. 2010 J Bacteriol 192: 111-116). Gene disruption analysis revealed that the spore coat protein CotB1 mediates the accumulation of silica (our unpublished results). Here, we report that B. cereus CotB1 (171 amino acids [aa]) and its C-terminal 14-aa region (corresponding to residues 158-171, designated CotB1p) show strong affinity for silica particles, with dissociation constants at pH 8.0 of 2.09 and 1.24 nM, respectively. Using CotB1 and CotB1p as silica-binding tags, we developed a silica-based affinity purification method in which silica particles are used as an adsorbent for CotB1/CotB1p fusion proteins. Small ubiquitin-like modifier (SUMO) technology was employed to release the target proteins from the adsorbed fusion proteins. SUMO-protease-mediated site-specific cleavage at the C-terminus of the fused SUMO sequence released the tagless target proteins into the liquid phase while leaving the tag region still bound to the solid phase. Using the fluorescent protein mCherry as a model, our purification method achieved 85 % recovery, with a purity of 95 % and yields of 0.60 ± 0.06 and 1.13 ± 0.13 mg per 10-mL bacterial culture for the CotB1-SUMO-mCherry and CotB1p-SUMO-mCherry fusions, respectively. CotB1p, a short 14-aa peptide, which demonstrates high affinity for silica, could be a promising fusion tag for both affinity purification and enzyme immobilization on silica supports. PMID:24756322

  4. Employing linear tetranuclear [Zn4(COO)4(OH)2] clusters as building subunits to construct a new Zn(II) coordination polymer with tunable luminescent properties

    NASA Astrophysics Data System (ADS)

    Li, Wu-Wu; Zhang, Zun-Ting

    2016-02-01

    A new Zn(II) coordination polymer, [Zn2(btc) (biimpy) (OH)]n (1 H3btc = 1,3,5-benzenetricarboxylic acid, biimpy = 2,6-bis(1-imdazoly)pyridine) has been successfully synthesized and characterized by elemental analysis, powder single crystal X-ray diffraction analyses. Compound 1 features a 3D framework employing linear tetranuclear [Zn4(COO)4(OH)2] cluster as building subunits. Topological analysis reveals it represents a (3,10)-connected structural topology by viewing btc3-, linear tetranuclear clusters and biimpy as 3-connected nodes, 10-connected nodes, linear linkers, respectively. Moreover, the thermal stability and luminescent property of compound 1 have been well investigated.

  5. Isostructural 1D coordination polymers of Zn(II), Cd(II) and Cu(II) with phenylpropynoic acid and DABCO as organic linkers

    NASA Astrophysics Data System (ADS)

    Saravanakumar, Rajendran; Varghese, Babu; Sankararaman, Sethuraman

    2014-11-01

    Using phenylpropynoic acid (PPA) and 1,4-diazabicyclo[2.2.2]octane (DABCO) as organic spacers, isostructural coordination polymers of Zn(II), Cd(II) and Cu(II) were synthesized by solvothermal method and structurally characterized using single crystal XRD, powder XRD, 13C CP-MAS NMR spectroscopy. Single crystal XRD data revealed four PPA units coordinating with two metal ions forming a paddle wheel secondary building unit (SBU). The paddle wheel units are connected through coordination of DABCO nitrogen to the metal centers from the axial positions leading to the formation of the 1D coordination polymers along the c axis. Intermolecular π stacking and Csbnd H…π interactions between the adjacent polymer chains convert the 1D coordination polymer into an interesting 3D network with the Csbnd H…π bonds running along the crystallographic a and b axes. Thermal and nitrogen adsorption studies of these coordination polymers are reported.

  6. Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

    PubMed

    Marchenko, N Iu; Marchenkov, V V; Kaĭsheva, A L; Kashparov, I A; Kotova, N V; Kaliman, P A; Semisotnov, G V

    2006-12-01

    The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions. PMID:17223789

  7. Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

    PubMed

    Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

    2012-01-01

    To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T(9)TKE(12) sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K(d) = 25±6 nM to K(d) = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K(d) = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν(as)(P-O) and ν(s)(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν(as)(UO(2))(2+) vibration (from 923 cm(-1) to 908 cm(-1)) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

  8. Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

    PubMed Central

    Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

    2012-01-01

    To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

  9. Renal protein reactivity and stability of antibiotic amphenicols: structure and affinity.

    PubMed

    Ding, Fei; Peng, Wei; Peng, Yu-Kui; Jiang, Yu-Ting

    2014-10-01

    In the present work, the molecular recognition of the oldest active amphenicols by the most popular renal carrier, lysozyme, was deciphered by using fluorescence, circular dichroism (CD) and molecular modeling at the molecular scale. Steady state fluorescence data showed that the recognition of amphenicol by lysozyme yields a static type of fluorescence quenching. This corroborates time-resolved fluorescence results that lysozyme-amphenicol adduct formation has a moderate affinity of 10(4) M(-1), and the driving forces were found to be chiefly hydrogen bonds, hydrophobic interactions and π stacking. Far-UV CD spectra confirmed that the spatial structure of lysozyme was slightly changed with a distinct reduction of α-helices in the presence of amphenicol, suggesting partial destabilization of the protein. Furthermore, via the extrinsic 8-anilino-1-naphthalenesulfonic acid fluorescence spectral properties and molecular modeling, one could see that the amphenicol binding site was situated at the deep crevice on the protein surface, and the ligand was also near to several crucial amino acid residues, such as Trp-62, Trp-63 and Arg-73. Simultaneously, contrastive studies of protein-amphenicols revealed clearly that some substituting groups, e.g. nitryl in the molecular structure of ligands, may be vitally important for the recognition activity of amphenicols with lysozyme. Due to the connection of amphenicols with fatal detrimental effects and because lysozyme has been applied as a drug carrier for proximal tubular targeting, the discussion herein is necessary for rational antibiotic use, development of safe antibiotics and particularly a better appraisal of the risks associated with human exposure to toxic agrochemicals. PMID:25016933

  10. Rapid Imaging of Latent Fingerprints Using Biocompatible Fluorescent Silica Nanoparticles.

    PubMed

    Kim, Young-Jae; Jung, Hak-Sung; Lim, Joohyun; Ryu, Seung-Jin; Lee, Jin-Kyu

    2016-08-16

    Fluorescent silica nanoparticles (FSNPs) are synthesized through the Stöber method by incorporating silane-modified organic dye molecules. The modified fluorescent organic dye molecule is able to be prepared by allylation and hydrosilylation reactions. The optical properties of as-prepared FSNPs are shown the similar optical properties of PR254A (allylated Pigment Red 254) and have outstanding photostability. The polyvinylpyrrolidone (PVP) is introduced onto the surface of FSNP to enhance the binding affinity of PVP-coated FSNP for latent fingerprints (LFPs) detection. The simple preparation and easy control of surface properties of FSNPs show potential as a fluorescent labeling material for enhanced latent fingerprint detection on hydrophilic and hydrophobic substrates in forensic science for individual identification. PMID:27452188

  11. Coverslip Cleaning and Functionalization for Total Internal Reflection Fluorescence Microscopy.

    PubMed

    Kudalkar, Emily M; Deng, Yi; Davis, Trisha N; Asbury, Charles L

    2016-01-01

    Total internal reflection fluorescence (TIRF) microscopy allows visualization of biological events at the single-molecule level by restricting excitation to a precise focal plane near the coverslip and eliminating out-of-focus fluorescence. The quality of TIRF imaging relies on a high signal-to-noise ratio and therefore it is imperative to prevent adherence of molecules to the glass coverslip. Nonspecific interactions can make it difficult to distinguish true binding events and may also interfere with accurate quantification of background noise. In addition, nonspecific binding of the fluorescently tagged protein will lower the effective working concentration, thereby altering values used to calculate affinity constants. To prevent spurious interactions, we thoroughly clean the surface of the coverslip and then functionalize the glass either by applying a layer of silane or by coating with a lipid bilayer. PMID:27140911

  12. Dimeric fluorescent energy transfer dyes comprising asymmetric cyanine azole-indolenine chromophores

    DOEpatents

    Glazer, Alexander N.; Benson, Scott C.

    1996-01-01

    Novel fluorescent DNA-staining dyes are provided combining asymmetric cyanine azole-indolenine dyes, which provide for strong DNA affinity, large Stokes shifts and emission in the red region of the spectrum. The dyes find particular application in gel electrophoresis and for labels which may be bound to a variety of compositions in a variety of contexts.

  13. Plasmonically amplified bioassay - Total internal reflection fluorescence vs. epifluorescence geometry.

    PubMed

    Hageneder, Simone; Bauch, Martin; Dostalek, Jakub

    2016-08-15

    This paper investigates plasmonic amplification in two commonly used optical configurations for fluorescence readout of bioassays - epifluorescence (EPF) and total internal reflection fluorescence (TIRF). The plasmonic amplification in the EPF configuration was implemented by using crossed gold diffraction grating and Kretschmann geometry of attenuated total reflection method (ATR) was employed in the TIRF configuration. Identical assay, surface architecture for analyte capture, and optics for the excitation, collection and detection of emitted fluorescence light intensity were used in both TIRF and EPF configurations. Simulations predict that the crossed gold diffraction grating (EPF) can amplify the fluorescence signal by a factor of 10(2) by the combination of surface plasmon-enhanced excitation and directional surface plasmon-coupled emission in the red part of spectrum. This factor is about order of magnitude higher than that predicted for the Kretschmann geometry (TIRF) which only took advantage of the surface plasmon-enhanced excitation. When applied for the readout of sandwich interleukin 6 (IL-6) immunoassay, the plasmonically amplified EPF geometry designed for Alexa Fluor 647 labels offered 4-times higher fluorescence signal intensity compared to TIRF. Interestingly, both geometries allowed reaching the same detection limit of 0.4pM despite of the difference in the fluorescence signal enhancement. This is attributed to inherently lower background of fluorescence signal for TIRF geometry compared to that for EPF which compensates for the weaker fluorescence signal enhancement. The analysis of the inflammation biomarker IL-6 in serum at medically relevant concentrations and the utilization of plasmonic amplification for the fluorescence measurement of kinetics of surface affinity reactions are demonstrated for both EPF and TIRF readout. PMID:27260457

  14. FRET enhanced fluorescent nanodiamonds.

    PubMed

    Fudala, Rafal; Raut, Sangram; Maliwal, Badri P; Zerda, T W; Gryczynski, Ignacy; Simanek, Eric; Borejdo, Julian; Rich, Ryan; Akopova, Irina; Gryczynski, Zygmunt

    2014-01-01

    Fluorescent nanodiamonds (FNDs) are one of the new and very promising biocompatible nanomaterials that can be used both as a fluorescence imaging agent and a highly versatile platform for controlled functionalization to target and deliver a wide spectrum of therapeutic agents. Among the remarkable fluorescence properties are excellent photostability, emission between 600-700nm, quantum yield of 1 and moderately long fluorescence lifetimes. However the low absorption cross section of fluorescent (N-V)(-) centers limits FNDs' brightness. In this work we show that an approach based on the Forster resonance energy transfer (FRET) may significantly enhance the fluorescence signal observed from a single ND. We demonstrate that organic dyes (fluorophores) attached to the FND surface can efficiently transfer the excitation energy to (N-V)(-) centers. Multiple dyes positioned in close proximity to the ND facile surface may serve as harvesting antennas transferring excitation energy to the fluorescent centers. We propose that, with the help of some of the functional groups present on the FND surface, we can either directly link flurophores or use scalable dendrimer chemistry to position many organic dyes at a calibrated distance. Also, the remaining multiple functional groups will be still available for particle targeting and drug delivery. This opens a new way for designing a new type of theranostics particles of ultrahigh brightness, high photostability, specific targeting, and high capacity for drug delivery. PMID:22394126

  15. Kinetics and thermodynamics of metal-binding to histone deacetylase 8

    PubMed Central

    Kim, Byungchul; Pithadia, Amit S; Fierke, Carol A

    2015-01-01

    Histone deacetylase 8 (HDAC8) was originally classified as a Zn(II)-dependent deacetylase on the basis of Zn(II)-dependent HDAC8 activity in vitro and illumination of a Zn(II) bound to the active site. However, in vitro measurements demonstrated that HDAC8 has higher activity with a bound Fe(II) than Zn(II), although Fe(II)-HDAC8 rapidly loses activity under aerobic conditions. These data suggest that in the cell HDAC8 could be activated by either Zn(II) or Fe(II). Here we detail the kinetics, thermodynamics, and selectivity of Zn(II) and Fe(II) binding to HDAC8. To this end, we have developed a fluorescence anisotropy assay using fluorescein-labeled suberoylanilide hydroxamic acid (fl-SAHA). fl-SAHA binds specifically to metal-bound HDAC8 with affinities comparable to SAHA. To measure the metal affinity of HDAC, metal binding was coupled to fl-SAHA and assayed from the observed change in anisotropy. The metal KD values for HDAC8 are significantly different, ranging from picomolar to micromolar for Zn(II) and Fe(II), respectively. Unexpectedly, the Fe(II) and Zn(II) dissociation rate constants from HDAC8 are comparable, koff ∼0.0006 s−1, suggesting that the apparent association rate constant for Fe(II) is slow (∼3 × 103 M−1 s−1). Furthermore, monovalent cations (K+ or Na+) that bind to HDAC8 decrease the dissociation rate constant of Zn(II) by ≥100-fold for K+ and ≥10-fold for Na+, suggesting a possible mechanism for regulating metal exchange in vivo. The HDAC8 metal affinities are comparable to the readily exchangeable Zn(II) and Fe(II) concentrations in cells, consistent with either or both metal cofactors activating HDAC8. PMID:25516458

  16. Uptake and distribution of fluorescently labeled cobalamin in neoplastic and healthy breast tissue

    NASA Astrophysics Data System (ADS)

    Cannon, Michelle J.; McGreevy, James M.; Holden, Joseph A.; West, Frederick G.; Grissom, Charles B.

    2000-05-01

    Fluorescent analogs of cobalamin (vitamin B12) have been developed as diagnostic markers of cancer cells. These compounds are recognized by transcobalamin, a cobalamin transport protein, with high affinity, as shown by surface plasmon resonance. The cellular sequestration and gross distribution of fluorescent cobalamin bioconjugates in breast tissue is being examined by epifluorescence microscopy. The distribution of each compound is being evaluated in proliferative and non-proliferative tissue, i.e. normal tissue and breast carcinoma. The results of preliminary studies suggest that fluorescent analogs of cobalamin may be a useful tool in therapeutic breast operations to define tumor margins and to distinguish neoplastic breast tissue from healthy breast tissue.

  17. A highly sensitive aptasensor for OTA detection based on hybridization chain reaction and fluorescent perylene probe.

    PubMed

    Wang, Bin; Wu, Yuanya; Chen, Yanfen; Weng, Bo; Xu, Liqun; Li, Changming

    2016-07-15

    An optical aptasensor was developed for ultrasensitive detection of ochratoxin A (OTA) based on hybridization chain reaction (HCR) amplification strategy and fluorescent perylene probe (PAPDI)/DNA composites. Dendritic DNA concatamers were synthesized by HCR strategy and modified on magnetic nanoparticles through aptamer as medium. A large amount of PAPDI probe aggregated under the induction of DNA concatamers and caused fluorescence quenching. In the presence of OTA, the PAPDI/DNA composites were released from magnetic nanoparticles due to the strong affinity between aptamer and OTA. In ethanol, PAPDI monomers disaggregated and produced strong fluorescence. The present method displays excellent sensitivity and selectivity towards OTA. PMID:26938491

  18. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  19. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  20. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-11-29

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  1. Fluorescent discharge lamp

    NASA Technical Reports Server (NTRS)

    Mukai, E.; Otsuka, H.; Nomi, K.; Honmo, I.

    1982-01-01

    A rapidly illuminating fluorescent lamp 1,200 mm long and 32.5 mm in diameter with an interior conducting strip which is compatible with conventional fixtures and ballasts is described. The fluorescent lamp is composed of a linear glass tube, electrodes sealed at both ends, mercury and raregas sealed in the glass tube, a fluorescent substance clad on the inner walls of the glass tube, and a clad conducting strip extending the entire length of the glass tube in the axial direction on the inner surface of the tube.

  2. Five new Zn(II) and Cd(II) coordination polymers constructed by 3,5-bis-oxyacetate-benzoic acid: Syntheses, crystal structures, network topologies and luminescent properties

    SciTech Connect

    Jiang Xianrong; Yuan Hongyan; Feng Yunlong

    2012-07-15

    Five Zn(II) and Cd(II) coordination polymers, [Zn{sub 2}(BOABA)(bpp)(OH)]{center_dot}0.5H{sub 2}O (1), [Cd{sub 3}(BOABA){sub 2}(bpp){sub 2}(H{sub 2}O){sub 6}]{center_dot}2H{sub 2}O (2), [Cd{sub 3}(BOABA){sub 2}(2,2 Prime -bipy){sub 3}(H{sub 2}O){sub 4}]{center_dot}5.5H{sub 2}O (3), [CdNa(BOABA)(H{sub 2}O)]{sub 2}{center_dot}H{sub 2}O (4) and [Cd{sub 2}(BOABA)(bimb)Cl(H{sub 2}O){sub 2}]{center_dot}H{sub 2}O (5) (H{sub 3}BOABA=3,5-bis-oxyacetate-benzoic acid, bpp=1,3-bi(4-pyridyl)propane, 2,2 Prime -bipy=2,2 Prime -bipyridine, bimb=1,4-bis(imidazol-1 Prime -yl)butane), have been solvothermally synthesized and characterized by single-crystal X-ray diffraction, elemental analyses, IR spectra and TG analyses. 1 is an uninodal 4-connected 2D square grid network based on binuclear zinc clusters. 2 is 2D wavelike layer structure and further linked by hydrogen bonds into the final 3D (5,6,6)-connected topology network. 3 is 3-connected 2D topology network and the 2,2 Prime -bipy ligands decorate in two different types. 4 is a (4,8)-connected 2D topology network with heterocaryotic {l_brace}Cd{sub 2}Na{sub 2}{r_brace} clusters and BOABA{sup 3-} ligands. 5 can be rationalized as a (3,10)-connected 3D topology network with tetranuclear {l_brace}Cd{sub 4}Cl{sub 2}{r_brace} clusters and BOABA{sup 3-} ligands. Meanwhile, photoluminescence studies revealed that these five coordination polymers display strong fluorescent emission bands in the solid state at room temperature. - Graphical abstract: Five new d{sup 10} metal(II) coordination polymers based on H{sub 3}BOABA ligand were obtained and characterized. They display different topological structures and luminescent properties. Highlights: Black-Right-Pointing-Pointer Five d{sup 10} metal(II) polymers based on 3,5-bis-oxyacetate-benzoic acid were obtained. Black-Right-Pointing-Pointer The polymers were structurally characterized by single-crystal X-ray diffraction. Black-Right-Pointing-Pointer Polymers 1-5 display different

  3. A Differential Dielectric Affinity Glucose Sensor

    PubMed Central

    Huang, Xian; Leduc, Charles; Ravussin, Yann; Li, Siqi; Davis, Erin; Song, Bing; Li, Dachao; Xu, Kexin; Accili, Domenico; Wang, Qian; Leibel, Rudolph; Lin, Qiao

    2013-01-01

    A continuous glucose monitor with a differential dielectric sensor implanted within the subcutaneous tissue that determines the glucose in the interstitial fluid is presented. The device, created using microelectromechanical systems (MEMS) technology, consists of sensing and reference modules that are identical in design and placed in close proximity. Each module contains a microchamber housing a pair of capacitive electrodes residing on the device substrate and embedded in a suspended, perforated polymer diaphragm. The microchambers, enclosed in semi-permeable membranes, are filled with either a polymer solution that has specific affinity to glucose or a glucose-insensitive reference solution. To accurately determine the glucose concentration, changes in the permittivity of the sensing and the reference solutions induced by changes in glucose concentration are measured differentially. In vitro characterization demonstrated the sensor capable of measuring glucose concentrations from 0 to 500 mg/dL with resolution and accuracy of ∼1.7 μg/dL and ∼1.74 mg/dL, respectively. In addition, device drift was reduced to 1.4% (uncontrolled environment) and 11% (5 °C of temperature variation) of that from non-differential measurements, indicating significant stability improvements. Preliminary animal testing demonstrated that the differential sensor accurately tracks glucose concentration in blood. This sensor can potentially be used clinically as a subcutaneously implanted continuous monitoring device in diabetic patients. PMID:24220675

  4. Affine gravity, Palatini formalism and charges

    NASA Astrophysics Data System (ADS)

    Katz, Joseph; Livshits, Gideon I.

    2011-12-01

    Affine gravity and the Palatini formalism contribute both to produce a simple and unique formula for calculating charges at spatial and null infinity for Lovelock type Lagrangians whose variational derivatives do not depend on second-order derivatives of the field components. The method is based on the covariant generalization due to Julia and Silva of the Regge-Teitelboim procedure that was used to define properly the mass in the classical formulation of Einstein's theory of gravity. Numerous applications reproduce standard results obtained by other secure but mostly specialized method like in ADM energy for asymptotically flat spacetimes and in Abbot and Deser for asymptotically de Sitter and anti-de Sitter spacetimes, both at spatial infinity. As a novel application we calculate the Bondi energy loss in five dimensional gravity, based on the asymptotic solution given by Tanabe et al. and obtain, as expected, the same result. We also give the for Einstein-Gauss-Bonnet gravity and find the superpotential for Lovelock theories of gravity when the number of dimensions tends to infinity with maximally symmetrical boundaries. The paper is written in standard component formalism.

  5. Ligand Affinities Estimated by Quantum Chemical Calculations.

    PubMed

    Söderhjelm, Pär; Kongsted, Jacob; Ryde, Ulf

    2010-05-11

    We present quantum chemical estimates of ligand-binding affinities performed, for the first time, at a level of theory for which there is a hope that dispersion and polarization effects are properly accounted for (MP2/cc-pVTZ) and at the same time effects of solvation, entropy, and sampling are included. We have studied the binding of seven biotin analogues to the avidin tetramer. The calculations have been performed by the recently developed PMISP approach (polarizable multipole interactions with supermolecular pairs), which treats electrostatic interactions by multipoles up to quadrupoles, induction by anisotropic polarizabilities, and nonclassical interactions (dispersion, exchange repulsion, etc.) by explicit quantum chemical calculations, using a fragmentation approach, except for long-range interactions that are treated by standard molecular-mechanics Lennard-Jones terms. In order to include effects of sampling, 10 snapshots from a molecular dynamics simulation are studied for each biotin analogue. Solvation energies are estimated by the polarized continuum model (PCM), coupled to the multipole-polarizability model. Entropy effects are estimated from vibrational frequencies, calculated at the molecular mechanics level. We encounter several problems, not previously discussed, illustrating that we are first to apply such a method. For example, the PCM model is, in the present implementation, questionable for large molecules, owing to the use of a surface definition that gives numerous small cavities in a protein. PMID:26615702

  6. Divalent cation affinity sites in Paramecium aurelia.

    PubMed

    Fisher, G; Kaneshiro, E S; Peters, P D

    1976-05-01

    Sites with high calcium affinity in Paramecium aurelia were identified by high calcium (5 mM) fixation and electron microscope methods. Electron-opaque deposits were observed on the cytoplasmic side of surface membranes, particularly at the basal regions of cilia and trichocyst-pellicle fusion sites. Deposits were also observed on some smooth cytomembranes, within the axoneme of cilia, and on basal bodies. The divalent cations, Mg2+, Mn2+, Sr2+, Ni2+, Ba2+, and Zn2+, could be substituted for Ca2+ in the procedure. Deposits were larger with 5 mM Sr2+. Ba2+, and Mn2+ at ciliary transverse plates and the terminal plate of basal bodies. Microprobe analysis showed that Ca and C1 were concentrated within deposits. In some analyses, S and P were detected in deposits. Also, microprobe analysis of 5 mM Mn2+-fixed P. aurelia showed that those deposits were enriched in Mn and C1 and sometimes enriched in P. Deposits were seen only when the ciliates were actively swimming at the time of fixation. Locomotory mutants having defective membrane Ca-gating mechanisms and ciliates fixed while exhibiting ciliary reversal showed no obvious differences in deposition pattern and intensity. Possible correlations between electron-opaque deposits and the locations of intramembranous particles seen by freeze-fracture studied, as well as sites where fibrillar material associate with membranes are considered. The possibility that the action sites of calcium and other divalent cations were identified is discussed. PMID:1262398

  7. Multiplexed protein profiling by sequential affinity capture.

    PubMed

    Ayoglu, Burcu; Birgersson, Elin; Mezger, Anja; Nilsson, Mats; Uhlén, Mathias; Nilsson, Peter; Schwenk, Jochen M

    2016-04-01

    Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off-target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi-automated sequential capture assay. This novel bead-based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read-out by a secondary capture bead array. We demonstrate in a proof-of-concept setting that target detection via two sequential affinity interactions reduced off-target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA-based signal amplification, and demonstrate the applicability of the dual capture bead-based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond. PMID:26935855

  8. Affinity of guanosine derivatives for polycytidylate revisited

    NASA Technical Reports Server (NTRS)

    Kanavarioti, A.; Hurley, T. B.; Baird, E. E.

    1995-01-01

    Evidence is presented for complexation of guanosine 5'-monophosphate 2-methylimidazolide (2-MeImpG) with polycytidylate (poly(C)) at pH 8.0 and 23 degrees C in the presence of 1.0 M NaCl2 and 0.2 M MgCl2 in water. The association of 2-MeImpG with poly(C) was investigated using UV-vis spectroscopy as well as by monitoring the kinetics of the nucleophilic substitution reaction of the imidazole moiety by amines. The results of both methods are consistent with moderately strong poly(C) 2-MeImpG complexation and the spectrophotometric measurements allowed the construction of a binding isotherm with a concentration of 2-MeImpG equal to 5.55 +/- 0.15 mM at half occupancy. UV spectroscopy was employed to establish the binding of other guanosine derivatives on poly(C). These derivatives are guanosine 5'-monophosphate (5'GMP), guanosine 5'-monophosphate imidazolide (ImpG), and guanosine 5'-monophosphate morpholidate (morpG). Within experimental error these guanosine derivatives exhibit the same affinity for poly(C) as 2-MeImpG.

  9. Banach frames in the affine synthesis problem

    NASA Astrophysics Data System (ADS)

    Terekhin, Pavel A.

    2009-10-01

    We consider the problem of representing functions f\\in L^p(\\mathbb R^d) by a series in elements of the affine system \\displaystyle \\psi_{j,k}(x)=\\lvert\\det a_j\\rvert^{1/2}\\psi(a_jx-bk), \\qquad j\\in\\mathbb N, \\quad k\\in\\mathbb Z^d. The corresponding representation theorems are established on the basis of the frame inequalities \\displaystyle A\\Vert g\\Vert _q\\le\\Vert\\{(g,\\psi_{j,k})\\}\\Vert _Y\\le B\\Vert g\\Vert _q for the Fourier coefficients \\displaystyle(g,\\psi_{j,k})=\\int_{\\mathbb R^d}g(x)\\psi_{j,k}(x)\\,dx of functions g\\in L^q(\\mathbb R^d), 1/p+1/q=1, where {\\Vert\\cdot\\Vert}_Y is the norm in some Banach space of number families \\{y_{j,k}\\} and 0 are constants. In particular, it is proved that if the integral of a function \\psi\\in L^1\\cap L^p(\\mathbb R^d), 1, is nonzero, so \\displaystyle\\int_{\\mathbb R^d}\\psi(x)\\,dx\

  10. FAST TRACK COMMUNICATION: Affine constellations without mutually unbiased counterparts

    NASA Astrophysics Data System (ADS)

    Weigert, Stefan; Durt, Thomas

    2010-10-01

    It has been conjectured that a complete set of mutually unbiased bases in a space of dimension d exists if and only if there is an affine plane of order d. We introduce affine constellations and compare their existence properties with those of mutually unbiased constellations. The observed discrepancies make a deeper relation between the two existence problems unlikely.

  11. Tending to Change: Toward a Situated Model of Affinity Spaces

    ERIC Educational Resources Information Center

    Bommarito, Dan

    2014-01-01

    The concept of affinity spaces, a theoretical construct used to analyze literate activity from a spatial perspective, has gained popularity among scholars of literacy studies and, particularly, video-game studies. This article seeks to expand current notions of affinity spaces by identifying key assumptions that have limited researchers'…

  12. The Study of Affinity-Seeking in an Organizational Setting.

    ERIC Educational Resources Information Center

    Flath, Dominic B.

    This study investigated the relationship between supervisors' use of Bell and Daly's affinity-seeking strategies and their impact on employee satisfaction. Results indicated that 16 of the 25 affinity-seeking strategies were positively correlated with a subordinate's perception of supervisor credibility. Results also indicated that a supervisor's…

  13. A minimax approach to spatial estimation using affinity matrices

    NASA Technical Reports Server (NTRS)

    Morris, C. N.

    1983-01-01

    Estimates made in the plane to improve on noisy unbiased estimates were combined. Only a small fraction of points in a giant grid were used to do this, those that are most like a given point. A component of this process defining an affinity matrix of values, indicating which points are relevant to others. Minimax rules are shown to be based on affinity matrices.

  14. Striving for Empathy: Affinities, Alliances and Peer Sexuality Educators

    ERIC Educational Resources Information Center

    Fields, Jessica; Copp, Martha

    2015-01-01

    Peer sexuality educators' accounts of their work reveal two approaches to empathy with their students: affinity and alliance. "Affinity-based empathy" rests on the idea that the more commonalities sexuality educators and students share (or perceive they share), the more they will be able to empathise with one another, while…

  15. Conformational kinetics reveals affinities of protein conformational states

    PubMed Central

    Daniels, Kyle G.; Suo, Yang; Oas, Terrence G.

    2015-01-01

    Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein’s affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states. PMID:26162682

  16. Affine group formulation of the Standard Model coupled to gravity

    SciTech Connect

    Chou, Ching-Yi; Ita, Eyo; Soo, Chopin

    2014-04-15

    In this work we apply the affine group formalism for four dimensional gravity of Lorentzian signature, which is based on Klauder’s affine algebraic program, to the formulation of the Hamiltonian constraint of the interaction of matter and all forces, including gravity with non-vanishing cosmological constant Λ, as an affine Lie algebra. We use the hermitian action of fermions coupled to gravitation and Yang–Mills theory to find the density weight one fermionic super-Hamiltonian constraint. This term, combined with the Yang–Mills and Higgs energy densities, are composed with York’s integrated time functional. The result, when combined with the imaginary part of the Chern–Simons functional Q, forms the affine commutation relation with the volume element V(x). Affine algebraic quantization of gravitation and matter on equal footing implies a fundamental uncertainty relation which is predicated upon a non-vanishing cosmological constant. -- Highlights: •Wheeler–DeWitt equation (WDW) quantized as affine algebra, realizing Klauder’s program. •WDW formulated for interaction of matter and all forces, including gravity, as affine algebra. •WDW features Hermitian generators in spite of fermionic content: Standard Model addressed. •Constructed a family of physical states for the full, coupled theory via affine coherent states. •Fundamental uncertainty relation, predicated on non-vanishing cosmological constant.

  17. Sulfation of Lower Chlorinated Polychlorinated Biphenyls Increases Their Affinity for the Major Drug-Binding Sites of Human Serum Albumin.

    PubMed

    Rodriguez, Eric A; Li, Xueshu; Lehmler, Hans-Joachim; Robertson, Larry W; Duffel, Michael W

    2016-05-17

    The disposition of toxicants is often affected by their binding to serum proteins, of which the most abundant in humans is serum albumin (HSA). There is increasing interest in the toxicities of environmentally persistent polychlorinated biphenyls (PCBs) with lower numbers of chlorine atoms (LC-PCBs) due to their presence in both indoor and outdoor air. PCB sulfates derived from metabolic hydroxylation and sulfation of LC-PCBs have been implicated in endocrine disruption due to high affinity-binding to the thyroxine-carrying protein, transthyretin. Interactions of these sulfated metabolites of LC-PCBs with HSA, however, have not been previously explored. We have now determined the relative HSA-binding affinities for a group of LC-PCBs and their hydroxylated and sulfated derivatives by selective displacement of the fluorescent probes 5-dimethylamino-1-naphthalenesulfonamide and dansyl-l-proline from the two major drug-binding sites on HSA (previously designated as Site I and Site II). Values for half-maximal displacement of the probes indicated that the relative binding affinities were generally PCB sulfate ≥ OH-PCB > PCB, although this affinity was site- and congener-selective. Moreover, specificity for Site II increased as the numbers of chlorine atoms increased. Thus, hydroxylation and sulfation of LC-PCBs result in selective interactions with HSA which may affect their overall retention and toxicity. PMID:27116425

  18. A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis.

    PubMed

    Enander, Karin; Dolphin, Gunnar T; Liedberg, Bo; Lundström, Ingemar; Baltzer, Lars

    2004-05-17

    A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target-protein binding. The platform is based on a versatile 42-residue helix-loop-helix polypeptide that dimerizes to form four-helix bundles and allows site-selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues. The well-characterized interaction between the model target-protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof-of-concept demonstration. An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold. The affinities of the array members for human carbonic anhydrase II (HCAII) were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant Kd=0.02-3 microM). The affinity of HCAII for acetazolamide (Kd=4 nM) was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target-protein binders. Also, successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform-expression profiling. Our platform is predicted to become a flexible tool for a variety of biosensor and protein-microarray applications within biochemistry, diagnostics and pharmaceutical chemistry. PMID:15146511

  19. Optimal T-cell receptor affinity for inducing autoimmunity.

    PubMed

    Koehli, Sabrina; Naeher, Dieter; Galati-Fournier, Virginie; Zehn, Dietmar; Palmer, Ed

    2014-12-01

    T-cell receptor affinity for self-antigen has an important role in establishing self-tolerance. Three transgenic mouse strains expressing antigens of variable affinity for the OVA transgenic-I T-cell receptor were generated to address how TCR affinity affects the efficiency of negative selection, the ability to prime an autoimmune response, and the elimination of the relevant target cell. Mice expressing antigens with an affinity just above the negative selection threshold exhibited the highest risk of developing experimental autoimmune diabetes. The data demonstrate that close to the affinity threshold for negative selection, sufficient numbers of self-reactive T cells escape deletion and create an increased risk for the development of autoimmunity. PMID:25411315

  20. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture. PMID:27473483

  1. Antimicrobial photodisinfection with Zn(II) phthalocyanine adsorbed on TiO2 upon UVA and red irradiation

    NASA Astrophysics Data System (ADS)

    Mantareva, Vanya; Eneva, Ivelina; Kussovski, Vesselin; Borisova, Ekaterina; Angelov, Ivan

    2015-01-01

    The light exposure on a daily basis has been well accepted as a competitive method for decontamination of wastewater. The catalytic properties of TiO2 offer a great potential to reduce the transmission of pathogens in the environment. Although the titanium dioxide shows high activity against pathogens, its general usage in water cleaning is limited due to the insufficient excitation natural light (about 3% of the solar spectrum). A hydrophobic dodecylpyridyloxy Zn(II)-phthalocyanine with four peripheral hydrocarbon chains of C12 (ZnPcDo) was immobilized on a photocatalyst TiO2 anatase (P25). The resulted greenish colored nanoparticles of phthalocyanine were characterized by the means of absorption, fluorescence and infrared spectroscopy. The laser scanning confocal fluorescence microscopy was used to visualize the phthalocyanine dye by the red fluorescence emission (650 - 740 nm). The intensive Q-band in the far red visible spectral region (~ 690 nm) suggested a monomeric state of phthalocyanine on TiO2 nanoparticles. Two pathogenic bacterial strains (methicillin-resistant Staphylococcus aureus - MRSA and Salmonella enteritidis) associated with wastewater were photoinactivated with the suspension of the particles. The effective photoinactivation was observed with 1 g.L-1 TiO2 anatase at irradiation with UVA 364 nm as with UVA 364 nm and LED 643 nm. The gram-negative Salmonella enteritidis was fully photoinactivated with ZnPcDo-TiO2 and TiO2 alone at UVA 346 nm and at irradiation with two light sources (364 nm + 643 nm). The proposed conjugate appears as an useful composite material for antibacterial disinfection.

  2. Fluorescent radiation converter

    NASA Technical Reports Server (NTRS)

    Viehmann, W. (Inventor)

    1981-01-01

    A fluorescence radiation converter is described which includes a substantially undoped optically transparent substrate and a waveshifter coating deposited on at least one portion of the substrate for absorption of radiation and conversion of fluorescent radiation. The coating is formed to substantially 1000 g/liter of a solvent, 70 to 200 g/liter of an organic polymer, and 0.2 to 25 g/liter of at least one organic fluorescent dye. The incoming incident radiation impinges on the coating. Radiation is absorbed by the fluorescent dye and is re-emitted as a longer wavelength radiation. Radiation is trapped within the substrate and is totally internally reflected by the boundary surface. Emitted radiation leaves the substrate ends to be detected.

  3. Fluorescent filtered electrophosphorescence

    DOEpatents

    Forrest, Stephen; Sun, Yiru; Giebink, Noel; Thompson, Mark E.

    2010-08-03

    The present invention relates to organic light emitting devices (OLEDs), and more specifically to OLEDS that emit light using a combination of fluorescent emitters and phosphorescent emitters for the efficient utilization of all of the electrically generated excitons.

  4. Fluorescent filtered electrophosphorescence

    DOEpatents

    Forrest, Stephen R.; Sun, Yiru; Giebink, Noel; Thompson, Mark E.

    2009-01-06

    The present invention relates to organic light emitting devices (OLEDs), and more specifically to OLEDS that emit light using a combination of fluorescent emitters and phosphorescent emitters for the efficient utilization of all of the electrically generated excitons.

  5. Fluorescent eye test (image)

    MedlinePlus

    The fluorescent eye test is useful in determining if there is a scratch or other problem with the surface ... has thoroughly covered the eye a cobalt blue light is then directed on the eye. The light ...

  6. Atmospheric Fluorescence Yield

    NASA Technical Reports Server (NTRS)

    Adams, James H., Jr.; Christl, M. J.; Fountain, W. F.; Gregory, J. C.; Martens, K.; Sokolsky, P.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    Several existing and planned experiments estimate the energies of ultra-high energy cosmic rays from air showers using the atmospheric fluorescence from these showers. Accurate knowledge of the conversion from atmospheric fluorescence to energy loss by ionizing particles in the atmosphere is key to this technique. In this paper we discuss a small balloon-borne instrument to make the first in situ measurements versus altitude of the atmospheric fluorescence yield. The instrument can also be used in the lab to investigate the dependence of the fluorescence yield in air on temperature, pressure and the concentrations of other gases that present in the atmosphere. The results can be used to explore environmental effects on and improve the accuracy of cosmic ray energy measurements for existing ground-based experiments and future space-based experiments.

  7. Chasing polys: Interdisciplinary affinity and its connection to physics identity

    NASA Astrophysics Data System (ADS)

    Scott, Tyler D.

    This research is based on two motivations that merge by means of the frameworks of interdisciplinary affinity and physics identity. First, a goal of education is to develop interdisciplinary abilities in students' thinking and work. But an often ignored factor is students interests and beliefs about being interdisciplinary. Thus, this work develops and uses a framework called interdisciplinary affinity. It encompasses students interests in making connections across disciplines and their beliefs about their abilities to make those connections. The second motivation of this research is to better understand how to engage more students with physics. Physics identity describes how a student sees themselves in relation to physics. By understanding how physics identity is developed, researchers and educators can identify factors that increase interest and engagement in physics classrooms. Therefore, physics identity was used in conjunction with interdisciplinary affinity. Using a mixed methods approach, this research used quantitative data to identify the relationships interdisciplinary affinity has with physics identity and the physics classroom. These connections were explored in more detail using a case study of three students in a high school physics class. Results showed significant and positive relationships between interdisciplinary affinity and physics identity, including the individual interest and recognition components of identity. It also identified characteristics of physics classrooms that had a significant, positive relationship with interdisciplinary affinity. The qualitative case study highlighted the importance of student interest to the relationship between interdisciplinary affinity and physics identity. It also identified interest and mastery orientation as key to understanding the link between interdisciplinary affinity and the physics classroom. These results are a positive sign that by understanding interdisciplinary affinity and physics identity

  8. Fluorescent Applications to Crystallization

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth; Achari, Aniruddha

    2006-01-01

    By covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and tests with model proteins have shown that labeling u to 5 percent of the protein molecules does not affect the X-ray data quality obtained . The presence of the trace fluorescent label gives a number of advantages. Since the label is covalently attached to the protein molecules, it "tracks" the protein s response to the crystallization conditions. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a darker background. Non-protein structures, such as salt crystals, do not show up under fluorescent illumination. Crystals have the highest protein concentration and are readily observed against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. Preliminary tests, using model proteins, indicates that we can use high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that more rapid amorphous precipitation kinetics may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Experiments are now being carried out to test this approach using a wider range, of proteins. The trace fluorescently labeled crystals will also

  9. Fluorescence endoscopic video system

    NASA Astrophysics Data System (ADS)

    Papayan, G. V.; Kang, Uk

    2006-10-01

    This paper describes a fluorescence endoscopic video system intended for the diagnosis of diseases of the internal organs. The system operates on the basis of two-channel recording of the video fluxes from a fluorescence channel and a reflected-light channel by means of a high-sensitivity monochrome television camera and a color camera, respectively. Examples are given of the application of the device in gastroenterology.

  10. Epi-Fluorescence Microscopy

    PubMed Central

    Webb, Donna J.; Brown, Claire M.

    2012-01-01

    Epi-fluorescence microscopy is available in most life sciences research laboratories, and when optimized can be a central laboratory tool. In this chapter, the epi-fluorescence light path is introduced and the various components are discussed in detail. Recommendations are made for incident lamp light sources, excitation and emission filters, dichroic mirrors, objective lenses, and charge-coupled device (CCD) cameras in order to obtain the most sensitive epi-fluorescence microscope. The even illumination of metal-halide lamps combined with new “hard” coated filters and mirrors, a high resolution monochrome CCD camera, and a high NA objective lens are all recommended for high resolution and high sensitivity fluorescence imaging. Recommendations are also made for multicolor imaging with the use of monochrome cameras, motorized filter turrets, individual filter cubes, and corresponding dyes that are the best choice for sensitive, high resolution multicolor imaging. Images should be collected using Nyquist sampling and should be corrected for background intensity contributions and nonuniform illumination across the field of view. Photostable fluorescent probes and proteins that absorb a lot of light (i.e., high extinction co-efficients) and generate a lot of fluorescence signal (i.e., high quantum yields) are optimal. A neuronal immune-fluorescence labeling protocol is also presented. Finally, in order to maximize the utility of sensitive wide-field microscopes and generate the highest resolution images with high signal-to-noise, advice for combining wide-field epi-fluorescence imaging with restorative image deconvolution is presented. PMID:23026996

  11. Spermine detection via metal-mediated ethynylarene ‘turn-on’ fluorescence signaling

    PubMed Central

    Fletcher, James T.; Bruck, Brent S.

    2014-01-01

    A dicarboxylated ethynylarene was shown to behave as a fluorescent chemosensor for millimolar concentrations of polyamines when mixed with Cd(II), Pb(II) or Zn(II) ions at micromolar concentrations. A bathochromic shift and intensification of fluorescence emission was observed with increasing amounts of metal ion in the presence of aqueous polyamines buffered at pH = 7.6. Such perturbations manifested as ‘turn-on’ signals from a ratiometric comparison of emission intensities at 390 nm versus 340 nm. Using Pb(II) as the metal mediator, spermine was selectively detected as a 40-fold signal enhancement relative to spermidine, putrescine, cadaverine and several other non-biogenic diamines. Evaluation of additional triamine and tetraamine analytes showed the influence that amine group quantity and spacing had on signal generation. By increasing the ratio of Pb(II) relative to ethynylarene, the detection limit for spermine was successfully lowered to a 25 micromolar level. Noncovalent association between ethynylarene, metal ion and polyamine are believed to promote the observed spectroscopic changes. This study exploits the subtle impact that polyamine structural identity has on transition metal chelation to define a new approach towards polyamine chemosensor development. PMID:25530671

  12. Screening Platform toward New Anti-HIV Aptamers Set on Molecular Docking and Fluorescence Quenching Techniques.

    PubMed

    Oliviero, Giorgia; Stornaiuolo, Mariano; D'Atri, Valentina; Nici, Fabrizia; Yousif, Ali Munaim; D'Errico, Stefano; Piccialli, Gennaro; Mayol, Luciano; Novellino, Ettore; Marinelli, Luciana; Grieco, Paolo; Carotenuto, Alfonso; Noppen, Sam; Liekens, Sandra; Balzarini, Jan; Borbone, Nicola

    2016-02-16

    By using a new rapid screening platform set on molecular docking simulations and fluorescence quenching techniques, three new anti-HIV aptamers targeting the viral surface glycoprotein 120 (gp120) were selected, synthesized, and assayed. The use of the short synthetic fluorescent peptide V35-Fluo mimicking the V3 loop of gp120, as the molecular target for fluorescence-quenching binding affinity studies, allowed one to measure the binding affinities of the new aptamers for the HIV-1 gp120 without the need to obtain and purify the full recombinant gp120 protein. The almost perfect correspondence between the calculated Kd and the experimental EC50 on HIV-infected cells confirmed the reliability of the platform as an alternative to the existing methods for aptamer selection and measuring of aptamer-protein equilibria. PMID:26810800

  13. Fluorogenic Enhancement of an in Vitro-Selected Peptide Ligand by Replacement of a Fluorescent Group.

    PubMed

    Wang, Wei; Zhu, Liping; Hirano, Yoshinori; Kariminavargani, Marziyeh; Tada, Seiichi; Zhang, Guanxin; Uzawa, Takanori; Zhang, Deqing; Hirose, Takuji; Taiji, Makoto; Ito, Yoshihiro

    2016-08-16

    To prepare a fluorogenic peptide ligand which binds to an arbitrary target, we previously succeeded in seeking a fluorogenic ligand to calmodulin using in vitro selection. In this study the environment-sensitive fluorescent group in the selected peptide ligand was replaced with other fluorescent groups to find the possibility to increase the fluorogenic activity. Surface plasmon resonance measurement exhibited that the binding affinity was held even after the replacement. However, the replacement significantly affected the fluorogenic activity. It depended on the kind of incorporated fluorophors and linker length. As a result, the incorporation of 4-N,N-dimethylamino-1,8-naphthalimide enhanced the fluorescence intensity over 100-fold in the presence of target calcium-bound calmodulin. This study demonstrated that the functionality of in vitro selected peptide can be tuned with keeping the binding affinity. PMID:27459509

  14. Clay-based affinity probes for selective cleanup and determination of aflatoxin B1 using nanostructured montmorillonite on quartz.

    PubMed

    Huebner, Henry J; Phillips, Timothy D

    2003-01-01

    A study was conducted to investigate the selective cleanup and determination of aflatoxin B1 (AfB1) from contaminated media. Composite adsorbents were formulated from calcium montmorillonite clay, which possesses a high affinity and enthalpy of adsorption for AfB1. Nanostructuring techniques were used to construct various formulations of the clay-based composite media. In AfB1 adsorption studies with prototypical affinity columns, these composites offered narrowly defined, reproducible capacity ranges. Composite recoveries of AfB1 from spiked grains exhibited linear trends that correlated well with the range of spike levels. Composite columns provided lower recoveries of AfB1 from naturally contaminated corn than did immunoaffinity columns; however, recoveries were consistent and purified extracts were free of interfering compounds, as determined by liquid chromatography with fluorescence detection. PMID:12852572

  15. Genetic affinities of central China populations.

    PubMed

    Zhou, H Y; Wang, H W; Tan, S N; Chen, Y; Wang, W L; Tao, H X; Yin, Z C; Zou, Y H; Ouyang, S M; Ni, B

    2014-01-01

    Hunan locates in the south-central part of China, to the south of the middle reaches of the Yangtze River and south of Lake Dongting. According to the historical records, the peopling of Hunan by modern human ancestors can ascend to 40 thousand years ago. Thus, to trace the ancient maternal components can offer further insight into the origin of south-central China. In this study, we investigated the mitochondrial DNA of 114 individuals from Hunan Province (including 34 Han, 40 Tujia and 40 Miao). Hypervariable regions I and II of the mtDNA control region were sequenced, and the relative diagnostic variations in coding region according to the updated worldwide phylogeny tree were selected and typed by restriction fragment length polymorphism analysis or direct sequencing. All individuals were classified into specific (sub)haplogroups. By comparison with the surrounding populations, southern China-prevalent haplogroups were detected with relative higher frequency in the Tujia and Miao ethnic populations, such as haplogroup B, with more than 20%, lacking in the Han population, which illustrated its southern origin characters. In addition, we also detected northern of East Asia prevalent haplogroups with a relative higher frequency in Tujia populations than in the Miao and Yao ethnic groups, implying a gene flow from Han populations. However, the language-clustering tendency was supported by our principal component analysis and further genetic estimation results. Han and ethnic groups in central China exhibited specific ancestors related to their closer language affinity, although there was extensively genetic admixture between Han and ethnic groups. PMID:24615027

  16. Synthesis, characterization, and biological activities of two Cu(II) and Zn(II) complexes with one polyquinoline ligand

    NASA Astrophysics Data System (ADS)

    Lu, Jing; Li, Jun-Ling; Sun, Qian; Jiang, Lin; Gu, Wen; Liu, Xin; Tian, Jin-Lei; Yan, Shi-Ping

    2014-09-01

    Two new complexes, [CuLCl]ClO4 (1) and [Zn2L2SO4(H2O)2](ClO4)2 (2) [L = N,N-bis(quinolin-2-ylmethyl)quinolin-8-amine], have been synthesized and structurally characterized. The interactions of two complexes with CT-DNA have been investigated by UV absorption, fluorescence spectroscopy, viscosity measurements and gel electrophoresis under physiological conditions. Results show that the complexes bind to CT-DNA with a moderate intercalative mode and exhibit efficient DNA cleavage activity on UV-A light of 365 nm. Furthermore, two complexes could quench the intrinsic fluorescence of BSA in a static quenching process based on BSA binding experiments. Notably, in vitro cytotoxicity study of two complexes on four human tumor cells lines (7404, HeLa, MCF-7, and HepG-2) indicate that both of them have the potential to act as effective anticancer drugs with low IC50 values.

  17. Selectively Promiscuous Opioid Ligands: Discovery of High Affinity/Low Efficacy Opioid Ligands with Substantial Nociceptin Opioid Peptide Receptor Affinity

    PubMed Central

    2015-01-01

    Emerging clinical and preclinical evidence suggests that a compound displaying high affinity for μ, κ, and δ opioid (MOP, KOP, and DOP) receptors and antagonist activity at each, coupled with moderate affinity and efficacy at nociceptin opioid peptide (NOP) receptors will have utility as a relapse prevention agent for multiple types of drug abuse. Members of the orvinol family of opioid ligands have the desired affinity profile but have typically displayed substantial efficacy at MOP and or KOP receptors. In this study it is shown that a phenyl ring analogue (1d) of buprenorphine displays the desired profile in vitro with high, nonselective affinity for the MOP, KOP, and DOP receptors coupled with moderate affinity for NOP receptors. In vivo, 1d lacked any opioid agonist activity and was an antagonist of both the MOP receptor agonist morphine and the KOP receptor agonist ethylketocyclazocine, confirming the desired opioid receptor profile in vivo. PMID:24761755

  18. Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Smith, Elizabeth Myhra

    The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

  19. Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na+ channel.

    PubMed

    Henry, Pauline C; Kanelis, Voula; O'Brien, M Christine; Kim, Brian; Gautschi, Ivan; Forman-Kay, Julie; Schild, Laurent; Rotin, Daniela

    2003-05-30

    The epithelial Na+ channel (alphabetagammaENaC) regulates salt and fluid homeostasis and blood pressure. Each ENaC subunit contains a PY motif (PPXY) that binds to the WW domains of Nedd4, a Hect family ubiquitin ligase containing 3-4 WW domains and usually a C2 domain. It has been proposed that Nedd4-2, but not Nedd4-1, isoforms can bind to and suppress ENaC activity. Here we challenge this notion and show that, instead, the presence of a unique WW domain (WW3*) in either Nedd4-2 or Nedd4-1 determines high affinity interactions and the ability to suppress ENaC. WW3* from either Nedd4-2 or Nedd4-1 binds ENaC-PY motifs equally well (e.g. Kd approximately 10 microm for alpha- or betaENaC, 3-6-fold higher affinity than WW4), as determined by intrinsic tryptophan fluorescence. Moreover, dNedd4-1, which naturally contains a WW3* instead of WW2, is able to suppress ENaC function equally well as Nedd4-2. Homology models of the WW3*.betaENaC-PY complex revealed that a Pro and Ala conserved in all WW3*, but not other Nedd4-WW domains, help form the binding pocket for PY motif prolines. Extensive contacts are formed between the betaENaC-PY motif and the Pro in WW3*, and the small Ala creates a large pocket to accommodate the peptide. Indeed, mutating the conserved Pro and Ala in WW3* reduces binding affinity 2-3-fold. Additionally, we demonstrate that mutations in PY motif residues that form contacts with the WW domain based on our previously solved structure either abolish or severely reduce binding affinity to the WW domain and that the extent of binding correlates with the level of ENaC suppression. Independently, we show that a peptide encompassing the PY motif of sgk1, previously proposed to bind to Nedd4-2 and alter its ability to regulate ENaC, does not bind (or binds poorly) the WW domains of Nedd4-2. Collectively, these results suggest that high affinity of WW domain-PY-motif interactions rather than affiliation with Nedd4-1/Nedd-2 is critical for ENaC suppression

  20. A new boronic acid fluorescent sensor based on fluorene for monosaccharides at physiological pH

    NASA Astrophysics Data System (ADS)

    Hosseinzadeh, Rahman; Mohadjerani, Maryam; Pooryousef, Mona; Eslami, Abbas; Emami, Saeed

    2015-06-01

    Fluorescent boronic acids are very useful fluorescent sensor for detection of biologically important saccharides. Herein we synthesized a new fluorene-based fluorescent boronic acid that shows significant fluorescence changes upon addition of saccharides at physiological pH. Upon addition of fructose, sorbitol, glucose, galactose, ribose, and maltose at different concentration to the solution of 7-(dimethylamino)-9,9-dimethyl-9H-fluoren-2-yl-2-boronic acid (7-DMAFBA, 1), significant decreases in fluorescent intensity were observed. It was found that this boronic acid has high affinity (Ka = 3582.88 M-1) and selectivity for fructose over glucose at pH = 7.4. The sensor 1 showed a linear response toward D-fructose in the concentrations ranging from 2.5 × 10-5 to 4 × 10-4 mol L-1 with the detection limit of 1.3 × 10-5 mol L-1.

  1. Fluorescent 2-Aminopyridine Nucleobases for Triplex-Forming Peptide Nucleic Acids.

    PubMed

    Cheruiyot, Samwel K; Rozners, Eriks

    2016-08-17

    Development of new fluorescent peptide nucleic acids (PNAs) is important for fundamental research and practical applications. The goal of this study was the design of fluorogenic nucleobases for incorporation in triplex-forming PNAs. The underlying design principle was the use of a protonation event that accompanied binding of a 2-aminopyridine (M) nucleobase to a G-C base pair as an on switch for a fluorescence signal. Two fluorogenic nucleobases, 3-(1-phenylethynyl)-M and phenylpyrrolo-M, were designed, synthesized and studied. The new M derivatives provided modest enhancement of fluorescence upon protonation but showed reduced RNA binding affinity and quenching of fluorescence signal upon triple-helix formation with cognate double-stranded RNA. Our study illustrates the principal challenges of design and provides guidelines for future improvement of fluorogenic PNA nucleobases. The 3-(1-phenylethynyl)-M may be used as a fluorescent nucleobase to study PNA-RNA triple-helix formation. PMID:27223320

  2. Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T Cells Bearing Extremely Low-Affinity TCRs

    PubMed Central

    Tungatt, Katie; Bianchi, Valentina; Crowther, Michael D.; Powell, Wendy E.; Schauenburg, Andrea J.; Trimby, Andrew; Donia, Marco; Miles, John J.; Holland, Christopher J.; Cole, David K.; Godkin, Andrew J.; Peakman, Mark; Straten, Per Thor; Svane, Inge Marie; Dolton, Garry

    2015-01-01

    Fluorochrome-conjugated peptide–MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II–restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR–pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents. PMID:25452566

  3. Tetrahydroprotoberberine alkaloids with dopamine and σ receptor affinity.

    PubMed

    Gadhiya, Satishkumar; Madapa, Sudharshan; Kurtzman, Thomas; Alberts, Ian L; Ramsey, Steven; Pillarsetty, Nagavara-Kishore; Kalidindi, Teja; Harding, Wayne W

    2016-05-01

    Two series of analogues of the tetrahydroprotoberberine (THPB) alkaloid (±)-stepholidine that (a) contain various alkoxy substituents at the C10 position and, (b) were de-rigidified with respect to (±)-stepholidine, were synthesized and evaluated for affinity at dopamine and σ receptors in order to evaluate effects on D3 and σ2 receptor affinity and selectivity. Small n-alkoxy groups are best tolerated by D3 and σ2 receptors. Among all compounds tested, C10 methoxy and ethoxy analogues (10 and 11 respectively) displayed the highest affinity for σ2 receptors as well as σ2 versus σ1 selectivity and also showed the highest D3 receptor affinity. De-rigidification of stepholidine resulted in decreased affinity at all receptors evaluated; thus the tetracyclic THPB framework is advantageous for affinity at dopamine and σ receptors. Docking of the C10 analogues at the D3 receptor, suggest that an ionic interaction between the protonated nitrogen atom and Asp110, a H-bond interaction between the C2 phenol and Ser192, a H-bond interaction between the C10 phenol and Cys181 as well as hydrophobic interactions of the aryl rings to Phe106 and Phe345, are critical for high affinity of the compounds. PMID:27032890

  4. Analysis of biomolecular interactions using affinity microcolumns: A review

    PubMed Central

    Zheng, Xiwei; Li, Zhao; Beeram, Sandya; Podariu, Maria; Matsuda, Ryan; Pfaunmiller, Erika L.; White, Christopher J.; Carter, NaTasha; Hage, David S.

    2014-01-01

    Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods. PMID:24572459

  5. Analysis of biomolecular interactions using affinity microcolumns: a review.

    PubMed

    Zheng, Xiwei; Li, Zhao; Beeram, Sandya; Podariu, Maria; Matsuda, Ryan; Pfaunmiller, Erika L; White, Christopher J; Carter, NaTasha; Hage, David S

    2014-10-01

    Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods. PMID:24572459

  6. Use of water-soluble PbS quantum dots as fluorescent probe in sensing copper(II)

    NASA Astrophysics Data System (ADS)

    Li, Ting; Wang, Nanxi; Chen, Lijia; Deng, Dawei; Gu, Yueqing

    2010-11-01

    In this paper, we report a new facile method for the synthesis of water-soluble PbS quantum dots (QDs), using dihydrolipoic acid (DHLA) as a stabilizer. The prepared QDs were characterized by optical techniques and high-resolution transmission electron microscopy (TEM). Next, these water-soluble luminescent PbS QDs were further used to detect copper (II). The obtained experimental results show that the fluorescence of the PbS QDs could be markedly quenched by Cu(II) whereas approximate concentrations of other physiologically relevant cations, such as Zn(II), Ca(II), Mg(II), Mn(II), Na(I) and K(I) etc., almost did not interfere with the fluorescence quenching progress of copper ions. Based on this, a simple and rapid method for Cu(II) determination was developed. Under optimal conditions, the response was linearly proportional to the copper(II) concentration in the range of 1 to 11.5x10-8 mol•L-1, with a correlation coefficient of 0.995. Hence, aqueous DHLA-stabilized PbS QDs may be a promising fluorescent probe in sensing copper(II) selectively.

  7. Metal-ion directed synthesis of N2O2 type chelate complexes of Ni(II), Cu(II) and Zn(II): spectral, thermal and single crystal studies.

    PubMed

    Yadav, Seema; Ahmad, Musheer; Siddiqi, K S

    2012-12-01

    A set of Ni(II), Cu(II) and Zn(II) complexes of Schiff base have been synthesized by one pot condensation reaction of 2-hydroxy-4-methoxybenzophenone with 1,3-diaminopropane in 1:2:1 molar ratio. They have been characterized by elemental analysis, conductance measurement, thermal analysis (TGA/DTA), FT-IR, (1)H-NMR, EPR spectra, ESI-MS and electronic spectral studies. In order to establish the geometry, a single crystal X-ray structure for [Ni(L)] complex was determined. It crystallizes in monoclinic system having unit cell parameters a=14.015(5)Å, b=13.391(5)Å, c=14.931(5)Å and α=90.000(5)(o), β=112.237(5)(o), γ=90.000(5)(o) with P 2(1)/c space group. No π-π stacking interaction was obtained in molecular packing diagram. A square-planar geometry has been confirmed for Ni(II) complex. Furthermore, on the basis of elemental analysis, IR, UV-visible and EPR data a square-planar geometry for Cu(II) and a distorted octahedral geometry for the Zn(II) complex has been established. In case of Zn(II) complex NO(3)(-) group was found to be coordinated in monodentate fashion. The low molar conductivity values of the complexes measured in chloroform and DMSO suggest their non-ionic nature. PMID:22960327

  8. Metal-ion directed synthesis of N2O2 type chelate complexes of Ni(II), Cu(II) and Zn(II): Spectral, thermal and single crystal studies

    NASA Astrophysics Data System (ADS)

    Yadav, Seema; Ahmad, Musheer; Siddiqi, K. S.

    2012-12-01

    A set of Ni(II), Cu(II) and Zn(II) complexes of Schiff base have been synthesized by one pot condensation reaction of 2-hydroxy-4-methoxybenzophenone with 1,3-diaminopropane in 1:2:1 molar ratio. They have been characterized by elemental analysis, conductance measurement, thermal analysis (TGA/DTA), FT-IR, 1H-NMR, EPR spectra, ESI-MS and electronic spectral studies. In order to establish the geometry, a single crystal X-ray structure for [Ni(L)] complex was determined. It crystallizes in monoclinic system having unit cell parameters a = 14.015(5) Å, b = 13.391(5) Å, c = 14.931(5) Å and α = 90.000(5)o, β = 112.237(5)o, γ = 90.000(5)o with P 21/c space group. No π-π stacking interaction was obtained in molecular packing diagram. A square-planar geometry has been confirmed for Ni(II) complex. Furthermore, on the basis of elemental analysis, IR, UV-visible and EPR data a square-planar geometry for Cu(II) and a distorted octahedral geometry for the Zn(II) complex has been established. In case of Zn(II) complex NO3- group was found to be coordinated in monodentate fashion. The low molar conductivity values of the complexes measured in chloroform and DMSO suggest their non-ionic nature.

  9. Highly selective removal of Zn(II) ion from hot-dip galvanizing pickling waste with amino-functionalized Fe3O4@SiO2 magnetic nano-adsorbent.

    PubMed

    Bao, Shuangyou; Tang, Lihong; Li, Kai; Ning, Ping; Peng, Jinhui; Guo, Huibin; Zhu, Tingting; Liu, Ye

    2016-01-15

    Amino-functionalized Fe3O4@SiO2 magnetic nano-adsorbent was used as a novel sorbent to highly selective removal of Zn(II) ion from hot-dip galvanizing pickling waste in the presence of Fe(II). These hot-dip galvanizing pickling waste mainly contain ZnCl2 and FeCl2 in aqueous HCl media. The properties of this magnetic adsorbent were examined by transmission electron microscopy (TEM), powder X-ray diffraction (XRD), infrared spectrometer (FT-IR) and BET surface area measurements. Various factors influencing the adsorption of Zn(II) ion such as initial concentration of metal ions, the amount of adsorbent, pH value of the solutions, the concentration of coexisting iron ion were investigated by batch experiments. The results indicated that the adsorption equilibrium data obeyed the Freundlich model with maximum adsorption capacities for Zn(II) to 169.5mg/g. The maximum adsorption occurred at pH 5±0.1 and Fe(II) interferences had no obvious influence. This work provides a potential and unique technique for zinc ion removal from hot-dip galvanizing pickling waste. PMID:26458121

  10. A fully-integrated aptamer-based affinity assay platform for monitoring astronaut health in space.

    SciTech Connect

    Yang, Xianbin; Durland, Ross H.; Hecht, Ariel H.; Singh, Anup K.; Sommer, Gregory Jon; Hatch, Anson V.

    2010-07-01

    Here we demonstrate the suitability of robust nucleic acid affinity reagents in an integrated point-of-care diagnostic platform for monitoring proteomic biomarkers indicative of astronaut health in spaceflight applications. A model thioaptamer targeting nuclear factor-kappa B (NF-{kappa}B) is evaluated in an on-chip electrophoretic gel-shift assay for human serum. Key steps of (i) mixing sample with the aptamer, (ii) buffer exchange, and (iii) preconcentration of sample were successfully integrated upstream of fluorescence-based detection. Challenges due to (i) nonspecific interactions with serum, and (ii) preconcentration at a nanoporous membrane are discussed and successfully resolved to yield a robust, rapid, and fully-integrated diagnostic system.

  11. Probing the Protein–Nanoparticle Interface: The Role of Aromatic Substitution Pattern on Affinity

    PubMed Central

    Ekmekci, Zeynep; Saha, Krishnendu; Moyano, Daniel F.; Tonga, Gulen Yesilbag; Wang, Hao; Mout, Rubul; Rotello, Vincent M.

    2016-01-01

    A new class of cationic gold nanoparticles has been synthesized bearing benzyl moieties featuring -NO2 and -OMe groups to investigate the regioisomeric control of aromatic nanoparticle-protein recognition. In general, nanoparticles bearing electron withdrawing group demonstrated higher binding affinities towards green fluorescent protein (GFP) compared to electron-donating groups. Significantly, a ~7.5 and ~4.3 fold increase in binding with GFP was observed for –NO2 groups in meta- and para-position respectively, while ortho-substitution showed similar binding compared to the unsubstituted ring. These findings demonstrated that nanoparticle-protein interaction can be controlled by the tuning the spatial orientation and the relative electronic properties of the aromatic substituents. This improved biomolecular recognition provides opportunities for enhanced biosensing and functional protein delivery to the cells. PMID:27122961

  12. New beginnings for matrix metalloproteinase inhibitors: identification of high-affinity zinc-binding groups.

    PubMed

    Puerta, David T; Lewis, Jana A; Cohen, Seth M

    2004-07-14

    In an effort to identify promising non-hydroxamate inhibitors of matrix metalloproteinases (MMPs), several new zinc-binding groups (ZBGs) based on pyrone, pyrothione, hydroxypyridinone, and hydroxypyridinethione chelators have been examined. Structural studies with tris(pyrazolyl)borate model complexes show that these ligands bind to the MMP active site zinc(II) ion in a bidentate fashion, similar to that found with hydroxamate-based inhibitors. Fluorescence- and colorimetric-based enzyme assays have been used to determine the IC50 values for these ZBGs against MMP-3; mixed O,S-donor ligands were found to be remarkably potent, with IC50 values as much as 700-fold lower than that found for acetohydroxamic acid. Inhibitory activity was found to parallel metal binding affinity as determined in titrations with model complexes. These results demonstrate that MPIs based on new ZBGs are feasible and may indeed improve the overall performance of inhibitors designed against these important medicinal targets. PMID:15237990

  13. ODE/IM correspondence and modified affine Toda field equations

    NASA Astrophysics Data System (ADS)

    Ito, Katsushi; Locke, Christopher

    2014-08-01

    We study the two-dimensional affine Toda field equations for affine Lie algebra gˆ modified by a conformal transformation and the associated linear equations. In the conformal limit, the associated linear problem reduces to a (pseudo-)differential equation. For classical affine Lie algebra gˆ, we obtain a (pseudo-)differential equation corresponding to the Bethe equations for the Langlands dual of the Lie algebra g, which were found by Dorey et al. in study of the ODE/IM correspondence.

  14. Strategies to guide the antibody affinity maturation process.

    PubMed

    Doria-Rose, Nicole A; Joyce, M Gordon

    2015-04-01

    Antibodies with protective activity are critical for vaccine efficacy. Affinity maturation increases antibody activity through multiple rounds of somatic hypermutation and selection in the germinal center. Identification of HIV-1 specific and influenza-specific antibody developmental pathways, as well as characterization of B cell and virus co-evolution in patients, has informed our understanding of antibody development. In order to counteract HIV-1 and influenza viral diversity, broadly neutralizing antibodies precisely target specific sites of vulnerability and require high levels of affinity maturation. We present immunization strategies that attempt to recapitulate these natural processes and guide the affinity maturation process. PMID:25913818

  15. Strategies to guide the antibody affinity maturation process

    PubMed Central

    Doria-Rose, Nicole A.; Joyce, M. Gordon

    2015-01-01

    Antibodies with protective activity are critical for vaccine efficacy. Affinity maturation increases antibody activity through multiple rounds of somatic hypermutation and selection in the germinal center. Identification of HIV-1 specific and influenza-specific antibody developmental pathways, as well as characterization of B cell and virus co-evolution in patients, has informed our understanding of antibody development. In order to counteract HIV-1 and Influenza viral diversity, broadly neutralizing antibodies precisely target specific sites of vulnerability and require high levels of affinity maturation. We present immunization strategies that attempt to recapitulate these natural processes and guide the affinity maturation process. PMID:25913818

  16. Affinity- and topology-dependent bound on current fluctuations

    NASA Astrophysics Data System (ADS)

    Pietzonka, Patrick; Barato, Andre C.; Seifert, Udo

    2016-08-01

    We provide a proof of a recently conjectured universal bound on current fluctuations in Markovian processes. This bound establishes a link between the fluctuations of an individual observable current, the cycle affinities driving the system into a non-equilibrium steady state, and the topology of the network. The proof is based on a decomposition of the network into independent cycles with both positive affinity and positive stationary cycle current. This formalism allows for a refinement of the bound for systems in equilibrium or with locally vanishing affinities.

  17. Affinity+: Semi-Structured Brainstorming on Large Displays

    SciTech Connect

    Burtner, Edwin R.; May, Richard A.; Scarberry, Randall E.; LaMothe, Ryan R.; Endert, Alexander

    2013-04-27

    Affinity diagraming is a powerful method for encouraging and capturing lateral thinking in a group environment. The Affinity+ Concept was designed to improve the collaborative brainstorm process through the use of large display surfaces in conjunction with mobile devices like smart phones and tablets. The system works by capturing the ideas digitally and allowing users to sort and group them on a large touch screen manually. Additionally, Affinity+ incorporates theme detection, topic clustering, and other processing algorithms that help bring structured analytic techniques to the process without requiring explicit leadership roles and other overhead typically involved in these activities.

  18. Fluorescent image tracking velocimeter

    DOEpatents

    Shaffer, Franklin D.

    1994-01-01

    A multiple-exposure fluorescent image tracking velocimeter (FITV) detects and measures the motion (trajectory, direction and velocity) of small particles close to light scattering surfaces. The small particles may follow the motion of a carrier medium such as a liquid, gas or multi-phase mixture, allowing the motion of the carrier medium to be observed, measured and recorded. The main components of the FITV include: (1) fluorescent particles; (2) a pulsed fluorescent excitation laser source; (3) an imaging camera; and (4) an image analyzer. FITV uses fluorescing particles excited by visible laser light to enhance particle image detectability near light scattering surfaces. The excitation laser light is filtered out before reaching the imaging camera allowing the fluoresced wavelengths emitted by the particles to be detected and recorded by the camera. FITV employs multiple exposures of a single camera image by pulsing the excitation laser light for producing a series of images of each particle along its trajectory. The time-lapsed image may be used to determine trajectory and velocity and the exposures may be coded to derive directional information.

  19. Protein-protein interaction studies based on molecular aptamers by affinity capillary electrophoresis.

    PubMed

    Huang, Chih-Ching; Cao, Zehui; Chang, Huan-Tsung; Tan, Weihong

    2004-12-01

    Protein-DNA/protein-protein interactions play critical roles in many biological processes. We report here the investigation of protein-protein interactions using molecular aptamers with affinity capillary electrophoresis (ACE). A human alpha-thrombin binding aptamer was labeled with 6-carboxyfluorescein and exploited as a selective fluorescent probe for studying thrombin-protein interactions using capillary electrophoresis with laser-induced fluorescence. A 15-mer binding DNA aptamer can be separated into two peaks in CE that correspond to the linear aptamer (L-Apt) and the thrombin-binding G-quadruplex structure in the presence of K(+) or Ba(2+). In a bare capillary, the peak area of G-quadruplex aptamer (G-Apt) was found to decrease with the addition of thrombin while that of L-Apt remained unchanged. Even though the peak of the G-Apt/thrombin binding complex is broad due to a weaker binding affinity between aptamer and thrombin, we were still able to quantify the thrombin and anti-thrombin proteins (human anti-thrombin III, AT III) based on the peak areas of free G-Apt. The detection limits of thrombin and AT III were 9.8 and 2.1 nM, respectively. The aptamer-based competitive ACE assay has also been applied to quantify thrombin-anti-thrombin III interaction and to monitor this reaction in real time. The addition of poly(ethylene glycol) to the sample matrix stabilized the complex of the G-Aptthrombin. This assay can be used to study the interactions between thrombin and proteins that do not disrupt G-Apt binding property at Exosit I site of the thrombin. Our aptamer-based ACE assay can be an effective approach for studying protein-protein interactions and for analyzing binding site and binding constant information in protein-protein and protein-DNA interaction studies. PMID:15571349

  20. Impact of D2 Receptor Internalization on Binding Affinity of Neuroimaging Radiotracers

    PubMed Central

    Guo, Ningning; Guo, Wen; Kralikova, Michaela; Jiang, Man; Schieren, Ira; Narendran, Raj; Slifstein, Mark; Abi-Dargham, Anissa; Laruelle, Marc; Javitch, Jonathan A; Rayport, Stephen

    2010-01-01

    Synaptic dopamine (DA) levels seem to affect the in vivo binding of many D2 receptor radioligands. Thus, release of endogenous DA induced by the administration of amphetamine decreases ligand binding, whereas DA depletion increases binding. This is generally thought to be due to competition between endogenous DA and the radioligands for D2 receptors. However, the temporal discrepancy between amphetamine-induced increases in DA as measured by microdialysis, which last on the order of 2 h, and the prolonged decrease in ligand binding, which lasts up to a day, has suggested that agonist-induced D2 receptor internalization may contribute to the sustained decrease in D2 receptor-binding potential seen following a DA surge. To test this hypothesis, we developed an in vitro system showing robust agonist-induced D2 receptor internalization following treatment with the agonist quinpirole. Human embryonic kidney 293 (HEK293) cells were stably co-transfected with human D2 receptor, G-protein-coupled receptor kinase 2 and arrestin 3. Agonist-induced D2 receptor internalization was demonstrated by fluorescence microscopy, flow cytometry, and radioligand competition binding. The binding of seven D2 antagonists and four agonists to the surface and internalized receptors was measured in intact cells. All the imaging ligands bound with high affinity to both surface and internalized D2 receptors. Affinity of most of the ligands to internalized receptors was modestly lower, indicating that internalization would reduce the binding potential measured in imaging studies carried out with these ligands. However, between-ligand differences in the magnitude of the internalization-associated affinity shift only partly accounted for the data obtained in neuroimaging experiments, suggesting the involvement of mechanisms beyond competition and internalization. PMID:19956086

  1. Vibriocholerae cytolysin recognizes the heptasaccharide core of complex N-glycans with nanomolar affinity

    PubMed Central

    Levan, Sophia; De, Swastik; Olson, Rich

    2013-01-01

    Pathogens selectively target host cells using adhesion molecules and secreted virulence factors that may utilize protein, lipid, or carbohydrate ligands on the cell surface. The human intestinal pathogen Vibrio cholerae secretes a pore-forming toxin, Vibrio cholerae cytolysin (VCC), which contains two domains that are structurally similar to known carbohydrate-binding proteins. These tandem domains are attached to the carboxy-terminus of the cytolytic domain and contain a β-trefoil fold and a β-prism fold. VCC has been shown to bind glycosylated proteins, and removal of the β-prism domain leads to a large decrease in lytic activity against rabbit erythrocytes. Despite these clues, the identity of the glycan receptors of VCC and the role of glycan binding in toxin activity remains unknown. To better understand this specificity, we used a combination of structural and functional approaches to characterize the carbohydrate-binding activity of the VCC toxin. We first probed the monosaccharide-binding activity of VCC and demonstrated that the toxin exhibits millimolar affinity for aldohexoses. To understand this specificity, we solved the crystal structure of the VCC β-prism domain bound to methyl-α-mannose. Next, we utilized a mammalian glycan screen to determine that the β-prism domain preferentially binds complex N-glycans with a heptasaccharide GlcNAc4 Man3 core (NGA2). Fluorescence anisotropy and surface plasmon resonance indicated an approximately 100-nanomolar affinity of the β-prism domain for the heptasaccharide core. Our results suggest that carbohydrate-binding domains on the VCC toxin facilitate high-affinity targeting of mammalian cell membranes, which may contribute to the ability of VCC to lyse cells at picomolar concentrations. PMID:23274141

  2. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  3. Fluorescent Exendin-4 Derivatives for Pancreatic β-Cell Analysis

    PubMed Central

    Clardy, Susan M.; Keliher, Edmund J.; Mohan, James F.; Sebas, Matt; Benoist, Christophe; Mathis, Diane; Weissleder, Ralph

    2014-01-01

    The ability to reliably identify pancreatic β-cells would have far reaching implications for a greater understanding of β-cell biology, measurement of β-cell mass in diabetes, islet transplantation, and drug development. The glucagon-like peptide-1 receptor (GLP1R) is highly expressed on the surface of insulin producing pancreatic β-cells. Using systematic modifications of the GLP1R ligand, exendin-4, we screened over 25 compounds and identified a palette of fluorescent exendin-4 with high GLP1R binding affinity. We show considerable differences in affinity, as well as utility of the top candidates for flow cytometry and microscopy of β-cells. Some of the developed compounds should be particularly useful for basic and translational β-cell research. PMID:24328216

  4. Metal binding affinity and structural properties of calmodulin-like protein 14 from Arabidopsis thaliana.

    PubMed

    Vallone, Rosario; La Verde, Valentina; D'Onofrio, Mariapina; Giorgetti, Alejandro; Dominici, Paola; Astegno, Alessandra

    2016-08-01

    In addition to the well-known Ca(2+) sensor calmodulin, plants possess many calmodulin-like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca(2+) and Mg(2+) binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS-based fluorescence, to evaluate the structural effects of metal binding and metal-induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca(2+) ion with micromolar affinity (Kd ∼ 12 µM) and the presence of 10 mM Mg(2+) decreases the Ca(2+) affinity by ∼5-fold. Although binding of Ca(2+) to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca(2+) sensors, but causes only localized structural changes in the unique functional EF-hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role. PMID:27124620

  5. Rapid identification of cholinesterase inhibitors from the seedcases of mangosteen using an enzyme affinity assay.

    PubMed

    Ryu, Hyung Won; Oh, Sei-Ryang; Curtis-Long, Marcus J; Lee, Ji Hye; Song, Hyuk-Hwan; Park, Ki Hun

    2014-02-12

    Enzyme binding affinity has been recently introduced as a selective screening method to identify bioactive substances within complex mixtures. We used an assay which identified small molecule binders of acetylcholinesterase (AChE) using the following series of steps: incubation of enzyme with extract; centrifugation and filtration; identification of small molecule content in the flow through. The crude extract contained 10 peaks in the UPLC chromatogram. However, after incubation the enzyme, six peaks were reduced, indicating these compounds bound AChE. All these isolated compounds (2, 3, and 5-8) significantly inhibited human AChE with IC₅₀s = 5.4-15.0 μM and butyrylcholinsterase (IC₅₀s = 0.7-11.0 μM). All compounds exhibited reversible mixed kinetics. Consistent with the binding screen and fluorescence quenching, γ-mangostin 6 had a much higher affinity for AChE than 9-hydroxycalabaxanthone 9. This validates this screening protocol as a rapid method to identify inhibitors of AChE. PMID:24446804

  6. Binding of angiogenesis inhibitor kringle 5 to its specific ligands by frontal affinity chromatography.

    PubMed

    Bian, Liujiao; Li, Qian; Ji, Xu

    2015-07-01

    The interactions between angiogenesis inhibitor Kringle 5 and its five specific ligands were investigated by frontal affinity chromatography in combination with fluorescence spectra and site-directed molecular docking. The binding constants of trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCHA), epsilon-aminocaproic acid (EACA), benzylamine, 7-aminoheptanoic acid (7-AHA) and L-lysine to Kringle 5 were 19.0×10(3), 7.97×10(3), 6.45×10(3), 6.07×10(3) and 4.04×10(3) L/mol, respectively. The five ligands bound to Kringle 5 on the lysine binding site in equimolar amounts, which was pushed mainly by hydrogen bond and Van der Waals force. This binding affinity was believed to be dependent on the functional group and flexible feature in ligands. This study will provide an important insight into the binding mechanism of angiogenesis inhibitor Kringle 5 to its specific ligands. PMID:25981289

  7. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  8. Eosinophil cationic protein high-affinity binding to bacteria-wall lipopolysaccharides and peptidoglycans.

    PubMed

    Torrent, Marc; Navarro, Susanna; Moussaoui, Mohammed; Nogués, M Victòria; Boix, Ester

    2008-03-18

    The eosinophil cationic protein (ECP) is an eosinophil-secreted RNase involved in the immune host defense, with a cytotoxic activity against a wide range of pathogens. The protein displays antimicrobial activity against both Gram-negative and Gram-positive strains. The protein can destabilize lipid bilayers, although the action at the membrane level can only partially account for its bactericidal activity. We have now shown that ECP can bind with high affinity to the bacteria-wall components. We have analyzed its specific association to lipopolysaccharides (LPSs), its lipid A component, and peptidoglycans (PGNs). ECP high-affinity binding capacity to LPSs and lipid A has been analyzed by a fluorescent displacement assay, and the corresponding dissociation constants were calculated using the protein labeled with a fluorophor. The protein also binds in vivo to bacteria cells. Ultrastructural analysis of cell bacteria wall and morphology have been visualized by scanning and transmission electron microscopy in both Escherichia coli and Staphylococcus aureus strains. The protein damages the bacteria surface and induces the cell population aggregation on E. coli cultures. Although both bacteria strain cells retain their shape and no cell lysis is patent, the protein can induce in E. coli the outer membrane detachment. ECP also activates the cytoplasmic membrane depolarization in both strains. Moreover, the depolarization activity on E. coli does not require any pretreatment to overcome the outer membrane barrier. The protein binding to the bacteria-wall surface would represent a first encounter step key in its antimicrobial mechanism of action. PMID:18293932

  9. "Smart" mobile affinity matrix for microfluidic immunoassays.

    PubMed

    Malmstadt, Noah; Hoffman, Allan S; Stayton, Patrick S

    2004-08-01

    There is a current need for simple methods for immobilizing biomolecules within microfluidic channels. Here, a technique is reported for reversibly immobilizing immunoassay components in a channel zone that can be simply controlled by integrated heating elements. Latex beads were modified with the temperature-responsive polymer poly(N-isopropylacrylamide)(PNIPAAm) and co-modified with biotinylated poly(ethylene glycol)(PEG). PNIPAAm undergoes a hydrophilic-to-hydrophobic transition when the temperature is raised above the lower critical solution temperature (LCST)( approximately 28 degrees C in the solutions used here). This reversible transition drives the aggregation and dis-aggregation of the modified beads in heated zones within poly(ethylene terephthalate)(PET) microchannels. Biotinylated monoclonal antibodies for the drug digoxin were bound via streptavidin to the biotin-PEG-coated beads. These antibody-functionalized beads were then reversibly immobilized by aggregation and hydrophobic adhesion to the surface of PET microfluidic channels in response to a thermal stimulus. The antibodies on the beads immobilized in the channel were shown to bind digoxin and a competitor fluorescent ligand from a flow stream in a quantitative competitive assay format that reported the digoxin concentration. The antibodies could be replenished for each immunoassay trial, using the reversible, temperature-controlled immobilization process. This technique allows reagent immobilization immediately prior to an analytical procedure, following the removal of previously utilized beads, guaranteeing fresh and active immobilized biomolecules. Furthermore, it provides a simple approach to multiplexing through the simultaneous or sequential injection of different antibody-coated bead species, potentially at multiple sites in the integrated device channels. PMID:15269814

  10. Relative DNA binding affinity of helix 3 homeodomain analogues, major groove binders, can be rapidly screened by displacement of prebound ethidium bromide. A comparative study.

    PubMed

    Shim, Yong-Ho; Arimondo, Paola B; Laigle, Alain; Garbesi, Anna; Lavielle, Solange

    2004-03-21

    The binding affinity for a 12-bp dsDNA of Antennapedia helix 3 analogues, major groove binders, has been measured by displacement of prebound ethidium bromide, a fluorescent displacement assay proposed for minor groove binders by Boger et al.(J. Am. Chem. Soc., 2000, 122, 6382-6394). Relative binding affinities determined by this method were compared to those obtained by gel mobility shift and footprinting assays for the 12-bp dsDNA and a 178-bp DNA fragment. The present work demonstrates that the fluorescence displacement assay is suitable for rapid screening of major groove binders, even though about 60 to 70% of the prebound ethidium bromide is displaced by these peptides. Total (100%) displacement of ethidium bromide was serendipitously achieved by addition in the peptide sequence, at the N-terminus, of a S-3-nitro-2-pyridinesulfenyl-N-acetyl-cysteine residue. S-3-nitro-2-pyridinesulfenylcysteine was shown to (i) bind to dsDNA with a micromolar affinity and (ii) direct within DNA grooves a peptide with no affinity for dsDNA. PMID:15007422

  11. Fiberized fluorescent dye microtubes

    NASA Astrophysics Data System (ADS)

    Vladev, Veselin; Eftimov, Tinko

    2013-03-01

    In the present work we study the effect of the length of fluorescent dye-filled micro-capillaries on the fluorescence spectra. Two types of micro-capillaries have been studied: a 100 μm inner diameter fused silica capillary with a transparent coating and one of the holes of a fiber optic glass ferrule with 125 μm inner diameter. The tubes were filled with solutions of Rhodamine 6G dissolved in ethanol and then in glycerin. Experimental data show that the maximum fluorescence and the largest spectral widths are observed for a sample length of about 0.25 mm for the used concentration. This results show that miniature tunable fiberized dye lasers can be developed using available standard micro-and fibre-optic components.

  12. Optically trapped fluorescent nanodiamonds

    NASA Astrophysics Data System (ADS)

    Horowitz, Viva R.; Alemán, Benjamin J.; Christle, David; Cleland, Andrew N.; Awschalom, David D.

    2012-02-01

    The electronic spin state of the nitrogen-vacancy (NV) center in diamond has gained considerable interest because it can be optically initialized, coherently manipulated, and optically read out at room temperature. In addition, nanoparticle diamonds containing NV centers can be integrated with biological and microfluidic systems. We have constructed and characterized an optical tweezers apparatus to trap fluorescent nanodiamonds in a fluid and measure their fluorescence. Particles are held and moved in three dimensions using an infrared trapping laser. Fluorescent detection of these optically trapped nanodiamonds enables us to observe nanoparticle dynamics and to measure electron spin resonance of NV centers. We will discuss applications using the electron spin resonance of trapped NV centers in nanodiamonds for magnetic field imaging in fluidic environments.

  13. Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble.

    PubMed

    Zandarashvili, Levani; Esadze, Alexandre; Vuzman, Dana; Kemme, Catherine A; Levy, Yaakov; Iwahara, Junji

    2015-09-15

    Although engineering of transcription factors and DNA-modifying enzymes has drawn substantial attention for artificial gene regulation and genome editing, most efforts focus on affinity and specificity of the DNA-binding proteins, typically overlooking the kinetic properties of these proteins. However, a simplistic pursuit of high affinity can lead to kinetically deficient proteins that spend too much time at nonspecific sites before reaching their targets on DNA. We demonstrate that structural dynamic knowledge of the DNA-scanning process allows for kinetically and thermodynamically balanced engineering of DNA-binding proteins. Our current study of the zinc-finger protein Egr-1 (also known as Zif268) and its nuclease derivatives reveals kinetic and thermodynamic roles of the dynamic conformational equilibrium between two modes during the DNA-scanning process: one mode suitable for search and the other for recognition. By mutagenesis, we were able to shift this equilibrium, as confirmed by NMR spectroscopy. Using fluorescence and biochemical assays as well as computational simulations, we analyzed how the shifts of the conformational equilibrium influence binding affinity, target search kinetics, and efficiency in displacing other proteins from the target sites. A shift toward the recognition mode caused an increase in affinity for DNA and a decrease in search efficiency. In contrast, a shift toward the search mode caused a decrease in affinity and an increase in search efficiency. This accelerated site-specific DNA cleavage by the zinc-finger nuclease, without enhancing off-target cleavage. Our study shows that appropriate modulation of the dynamic conformational ensemble can greatly improve zinc-finger technology, which has used Egr-1 (Zif268) as a major scaffold for engineering. PMID:26324943

  14. Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble

    PubMed Central

    Zandarashvili, Levani; Esadze, Alexandre; Vuzman, Dana; Kemme, Catherine A.; Levy, Yaakov; Iwahara, Junji

    2015-01-01

    Although engineering of transcription factors and DNA-modifying enzymes has drawn substantial attention for artificial gene regulation and genome editing, most efforts focus on affinity and specificity of the DNA-binding proteins, typically overlooking the kinetic properties of these proteins. However, a simplistic pursuit of high affinity can lead to kinetically deficient proteins that spend too much time at nonspecific sites before reaching their targets on DNA. We demonstrate that structural dynamic knowledge of the DNA-scanning process allows for kinetically and thermodynamically balanced engineering of DNA-binding proteins. Our current study of the zinc-finger protein Egr-1 (also known as Zif268) and its nuclease derivatives reveals kinetic and thermodynamic roles of the dynamic conformational equilibrium between two modes during the DNA-scanning process: one mode suitable for search and the other for recognition. By mutagenesis, we were able to shift this equilibrium, as confirmed by NMR spectroscopy. Using fluorescence and biochemical assays as well as computational simulations, we analyzed how the shifts of the conformational equilibrium influence binding affinity, target search kinetics, and efficiency in displacing other proteins from the target sites. A shift toward the recognition mode caused an increase in affinity for DNA and a decrease in search efficiency. In contrast, a shift toward the search mode caused a decrease in affinity and an increase in search efficiency. This accelerated site-specific DNA cleavage by the zinc-finger nuclease, without enhancing off-target cleavage. Our study shows that appropriate modulation of the dynamic conformational ensemble can greatly improve zinc-finger technology, which has used Egr-1 (Zif268) as a major scaffold for engineering. PMID:26324943

  15. Au25(SG)18 as a fluorescent iodide sensor

    NASA Astrophysics Data System (ADS)

    Wang, Man; Wu, Zhikun; Yang, Jiao; Wang, Guozhong; Wang, Hongzhi; Cai, Weiping

    2012-06-01

    The recently emerging gold nanoclusters (GNC) are of major importance for both basic science studies and practical applications. Based on its surface-induced fluorescence properties, we investigated the potential use of Au25(SG)18 (GSH: glutathione) as a fluorescent iodide sensor. The current detection limit of 400 nM, which can possibly be further enhanced by optimizing the conditions, and excellent selectivity among 12 types of anion (F-, Cl-, Br-, I-, NO3-, ClO4-, HCO3-, IO3-, SO42-, SO32-, CH3COO- and C6H5O73-) make Au25(SG)18 a good candidate for iodide sensing. Furthermore, our work has revealed the particular sensing mechanism, which was found to be affinity-induced ratiometric and enhanced fluorescence (abbreviated to AIREF), which has rarely been reported previously and may provide an alternative strategy for devising nanoparticle-based sensors.The recently emerging gold nanoclusters (GNC) are of major importance for both basic science studies and practical applications. Based on its surface-induced fluorescence properties, we investigated the potential use of Au25(SG)18 (GSH: glutathione) as a fluorescent iodide sensor. The current detection limit of 400 nM, which can possibly be further enhanced by optimizing the conditions, and excellent selectivity among 12 types of anion (F-, Cl-, Br-, I-, NO3-, ClO4-, HCO3-, IO3-, SO42-, SO32-, CH3COO- and C6H5O73-) make Au25(SG)18 a good candidate for iodide sensing. Furthermore, our work has revealed the particular sensing mechanism, which was found to be affinity-induced ratiometric and enhanced fluorescence (abbreviated to AIREF), which has rarely been reported previously and may provide an alternative strategy for devising nanoparticle-based sensors. Electronic supplementary information (ESI) available: fluorescence spectra of Au25(SG)18 (1.6 μM in H2O) with successive titration of I- and the time-dependent fluorescence of Au25(SG)18. See DOI: 10.1039/c2nr30169e.

  16. Extremely high negative electron affinity of diamond via magnesium adsorption

    NASA Astrophysics Data System (ADS)

    O'Donnell, K. M.; Edmonds, M. T.; Tadich, A.; Thomsen, L.; Stacey, A.; Schenk, A.; Pakes, C. I.; Ley, L.

    2015-07-01

    We report large negative electron affinity (NEA) on diamond (100) using magnesium adsorption on a previously oxygen-terminated surface. The measured NEA is up to (-2.01 ±0.05 ) eV, the largest reported negative electron affinity to date. Despite the expected close relationship between the surface chemistry of Mg and Li species on oxygen-terminated diamond, we observe differences in the adsorption properties between the two. Most importantly, a high-temperature annealing step is not required to activate the Mg-adsorbed surface to a state of negative electron affinity. Diamond surfaces prepared by this procedure continue to possess negative electron affinity after exposure to high temperatures, air, and even immersion in water.

  17. COMPARATIVE OXYGEN AFFINITY OF FISH AND MAMMALIAN MYOGLOBINS

    EPA Science Inventory

    Myoglobins from rat, coho salmon (Oncorhynchus kisutch), buffalo sculpin (Enophrys bison) hearts, and yellowfin tuna (Thunnus albacares) red skeletal muscle were partially purified and their O2 binding affinities determined. Commercially prepared sperm whale myoglobin was employe...

  18. Proton affinity of several basic non-standard amino acids

    NASA Astrophysics Data System (ADS)

    Rožman, Marko

    2012-08-01

    The structures and absolute proton affinities of several arginine (2-amino-3-guanidinopropionic acid, 2-amino-4-guanidinobutyric acid, homoarginine, citrulline and canavanine), histidine (1-methylhistidine and 3-methylhistidine) and lysine (2,3-diaminopropanoic acid, 2,4-diaminobutanoic acid, ornithine, 5-hydroxylysine, canaline and thialysine) homologues and analogues have been estimated using composite G3MP2B3 computational protocol. For a majority of here studied non-standard amino acids the gas-phase proton affinities were established for the first time, while for the others obtained values are used to improve the accuracy of the computational and experimental proton affinities reported previously. In addition, structures and proton affinities are discussed in order to rationalize their biological activity.

  19. Bidirectional Elastic Image Registration Using B-Spline Affine Transformation

    PubMed Central

    Gu, Suicheng; Meng, Xin; Sciurba, Frank C.; Wang, Chen; Kaminski, Naftali; Pu, Jiantao

    2014-01-01

    A registration scheme termed as B-spline affine transformation (BSAT) is presented in this study to elastically align two images. We define an affine transformation instead of the traditional translation at each control point. Mathematically, BSAT is a generalized form of the affine transformation and the traditional B-Spline transformation (BST). In order to improve the performance of the iterative closest point (ICP) method in registering two homologous shapes but with large deformation, a bi-directional instead of the traditional unidirectional objective / cost function is proposed. In implementation, the objective function is formulated as a sparse linear equation problem, and a sub-division strategy is used to achieve a reasonable efficiency in registration. The performance of the developed scheme was assessed using both two-dimensional (2D) synthesized dataset and three-dimensional (3D) volumetric computed tomography (CT) data. Our experiments showed that the proposed B-spline affine model could obtain reasonable registration accuracy. PMID:24530210

  20. Bidirectional elastic image registration using B-spline affine transformation.

    PubMed

    Gu, Suicheng; Meng, Xin; Sciurba, Frank C; Ma, Hongxia; Leader, Joseph; Kaminski, Naftali; Gur, David; Pu, Jiantao

    2014-06-01

    A registration scheme termed as B-spline affine transformation (BSAT) is presented in this study to elastically align two images. We define an affine transformation instead of the traditional translation at each control point. Mathematically, BSAT is a generalized form of the affine transformation and the traditional B-spline transformation (BST). In order to improve the performance of the iterative closest point (ICP) method in registering two homologous shapes but with large deformation, a bidirectional instead of the traditional unidirectional objective/cost function is proposed. In implementation, the objective function is formulated as a sparse linear equation problem, and a sub-division strategy is used to achieve a reasonable efficiency in registration. The performance of the developed scheme was assessed using both two-dimensional (2D) synthesized dataset and three-dimensional (3D) volumetric computed tomography (CT) data. Our experiments showed that the proposed B-spline affine model could obtain reasonable registration accuracy. PMID:24530210

  1. Frontal affinity chromatography (FAC): theory and basic aspects.

    PubMed

    Kasai, Ken-ichi

    2014-01-01

    Frontal affinity chromatography (FAC) is a versatile analytical tool for determining specific interactions between biomolecules and is particularly useful in the field of glycobiology. This article presents its basic aspects, merits, and theory. PMID:25117240

  2. Antibody Affinity Maturation in Fishes—Our Current Understanding

    PubMed Central

    Magor, Brad G.

    2015-01-01

    It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig) mutator enzyme activation-induced cytidine deaminase (AID). We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes. PMID:26264036

  3. Smartphone fluorescence spectroscopy.

    PubMed

    Yu, Hojeong; Tan, Yafang; Cunningham, Brian T

    2014-09-01

    We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of specific nucleic acid sequences in a liquid test sample and compared performance against a conventional laboratory fluorimeter. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route toward portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins. PMID:25098859

  4. Fast fluorescence holographic microscopy

    PubMed Central

    Qin, Wan; Yang, Xiaoqi; Li, Yingying; Peng, Xiang; Qu, Xinghua; Yao, Hai; Gao, Bruce Z.

    2015-01-01

    FINCHSCOPE is a new technology of fluorescence holographic microscopy. It has been successfully applied to recording high-resolution three-dimensional fluorescence images of biological specimens without the need for scanning. In this study, we revealed and analyzed an intrinsic phenomenon, called ghost lens effect, on spatial light modulator which is the core element enabling the incoherent correlation in the FINCHSCOPE. The ghost lens effect can degrade the imaging quality by introducing multiple spherical waves with different focal lengths into the correlation and thus increasing the noise in the recorded holograms. PMID:25767693

  5. Fluorescence activated cell sorting.

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Hulett, H. R.; Sweet, R. G.; Herzenberg, L. A.

    1972-01-01

    An instrument has been developed for sorting biological cells. The cells are rendered differentially fluorescent and incorporated into a small liquid stream illuminated by a laser beam. The cells pass sequentially through the beam, and fluorescent light from the cells gives rise to electrical signals. The stream is broken into a series of uniform size drops downstream of the laser. The cell signals are used to give appropriate electrostatic charges to drops containing the cells. The drops then pass between two charged plates and are deflected to appropriate containers. The system has proved capable of providing fractions containing large numbers of viable cells highly enriched in a particular functional type.

  6. Smartphone fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Yu, Hojoeng; Tan, Yafang; Cunningham, Brian T.

    2014-03-01

    We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of a specific nucleic acid sequences in a liquid test sample. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route towards portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.

  7. Nanosecond fluorescence spectroscopy

    SciTech Connect

    Leskovar, B.

    1985-03-01

    This article is a summary of a short course lecture given in conjunction with the 1984 Nuclear Science Symposium. Measuring systems for nanosecond fluorescence spectroscopy using single-photon counting techniques are presented. These involve systems based on relaxation-type spark gap light pulser and synchronously pumped mode-locked dye lasers. Furthermore, typical characteristics and optimization of operating conditions of the critical components responsible for the system time resolution are discussed. A short comparison of the most important deconvolution methods for numerical analysis of experimental data is given particularly with respect to the signal-to-noise ratio of the fluorescence signal. 22 refs., 8 figs.

  8. Aptamer-modified magnetic beads in affinity separation of proteins.

    PubMed

    Zhu, Guohong; Walter, Johanna-Gabriela

    2015-01-01

    Aptamers are valuable alternative ligands for affinity separations. Here, we describe the aptamer-based affinity separation of His-tagged proteins using an aptamer directed against the His-tag. The immobilization of the aptamer to magnetic beads is described as well as the aptamer-based purification and proper methods for the characterization of the process. Moreover, indications for the transfer of the process to other aptamers are given. PMID:25749947

  9. Detection of saccharides with a fluorescent sensing device based on a gold film modified with 4-mercaptophenylboronic acid monolayer

    NASA Astrophysics Data System (ADS)

    Chen, Shu-Jen; Chang, Jui-Feng; Cheng, Nai-Jen; Yih, Jeng-Nan; Chiu, Kuo-Chi

    2013-09-01

    An extremely sensitive fluorescent sensor based on a phenylboronic acid monolayer was developed for detecting saccharide molecules. The fluorescent sensor was prepared by assembling a monolayer of 4-mercaptophenylboronic acid (4-MPBA) onto a gold-coated compact disk. The change in the fluorescence of the 4-MPBA monolayer was extremely obvious in basic methanolic buffer containing monosaccharides down to the picomolar level. The fluorescence spectra demonstrated that the 4-MPBA monolayer was sensitive to monosaccharides and disaccharides, and the affinity of the monolayer toward saccharides was in the order of glucose < fructose < mannose < galactose < maltose > lactose > sucrose. Additionally, the fluorescence intensity of 4-MPBA monolayer was restorable after cleaning with weak acid, indicating that the reported fluorescent sensor with the detection limit of glucose down to the picomolar level is reusable for sensing saccharides.

  10. Proton affinity of methyl nitrate - Less than proton affinity of nitric acid

    NASA Technical Reports Server (NTRS)

    Lee, Timothy J.; Rice, Julia E.

    1992-01-01

    Several state-of-the-art ab initio quantum mechanical methods were used to investigate the equilibrium structure, dipole moments, harmonic vibrational frequencies, and IR intensities of methyl nitrate, methanol, and several structures of protonated methyl nitrate, using the same theoretical methods as in an earlier study (Lee and Rice, 1992) of nitric acid. The ab initio results for methyl nitrate and methanol were found to be in good agreement with available experimental data. The proton affinity (PA) of methyl nitrate was calculated to be 176.9 +/-5 kcal/mol, in excellent agreement with the experimental value 176 kcal/mol obtained by Attina et al. (1987) and less than the PA value of nitric acid. An explanation of the discrepancy of the present results with those of an earlier study on protonated nitric acid is proposed.

  11. Enhancing IHE XDS for federated clinical affinity domain support.

    PubMed

    Dogac, Asuman; Laleci, Gokce B; Aden, Thomas; Eichelberg, Marco

    2007-03-01

    One of the key problems in healthcare informatics is the inability to share patient records across enterprises. To address this problem, an important industry initiative called "integrating the healthcare enterprise (IHE)" specified the "cross enterprise document sharing (XDS)" profile. In the IHE XDS, healthcare enterprises that agree to work together form a "clinical affinity domain" and store healthcare documents in an ebXML registry/repository architecture to facilitate their sharing. The affinity domains also agree on a common set of policies such as coding lists to be used to annotate clinical documents in the registry/repository and the common schemes for patient identification. However, since patients expect their records to follow them as they move from one clinical affinity domain to another, there is a need for affinity domains to be federated to enable information exchange. In this paper, we describe how IHE XDS can be enhanced to support federated clinical affinity domains. We demonstrate that federation of affinity domains are facilitated when ontologies, rather than coding term lists, are used to annotate clinical documents. Furthermore, we describe a patient identification protocol that eliminates the need to keep a master patient index file for the federation. PMID:17390991

  12. Flexible Linker Modulates Glycosaminoglycan Affinity of Decorin Binding Protein A.

    PubMed

    Morgan, Ashli; Sepuru, Krishna Mohan; Feng, Wei; Rajarathnam, Krishna; Wang, Xu

    2015-08-18

    Decorin binding protein A (DBPA) is a glycosaminoglycan (GAG)-binding adhesin found on the surface of the bacterium Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease. DBPA facilitates bacterial adherence to extracellular matrices of human tissues and is crucial during the early stage of the infection process. Interestingly, DBPA from different strains (B31, N40, and PBr) show significant differences in GAG affinities, but the structural basis for the differences is not clear. In this study, we show that GAG affinity of N40 DBPA is modulated in part by flexible segments that control access to the GAG binding site, such that shortening of the linker leads to higher GAG affinity when analyzed using ELISA, gel mobility shift assay, solution NMR, and isothermal titration calorimetry. Our observation that GAG affinity differences among different B. burgdorferi strains can be attributed to a flexible linker domain regulating access to the GAG-binding domain is novel. It also provides a rare example of how neutral amino acids and dynamic segments in GAG binding proteins can have a large influence on GAG affinity and provides insights into why the number of basic amino acids in the GAG-binding site may not be the only factor determining GAG affinity of proteins. PMID:26223367

  13. ZCF32, a fungus specific Zn(II)2 Cys6 transcription factor, is a repressor of the biofilm development in the human pathogen Candida albicans.

    PubMed

    Kakade, Pallavi; Sadhale, Parag; Sanyal, Kaustuv; Nagaraja, Valakunja

    2016-01-01

    As a human fungal pathogen, Candida albicans can cause a wide variety of disease conditions ranging from superficial to systemic infections. Many of these infections are caused by an inherent ability of the pathogen to form biofilms on medical devices resulting in high mortality. Biofilms formed by C. albicans are a complex consortium of yeast and hyphal cells embedded in an extracellular matrix and are regulated by a network of transcription factors. Here, we report the role of a novel Zn(II)2-Cys6 binuclear cluster transcription factor, ZCF32, in the regulation of biofilm formation. Global transcriptome analysis reveals that biofilm development is the most altered pathway in the zcf32 null mutant. To delineate the functional correlation between ZCF32 and biofilm development, we determined the set of genes directly regulated by Zcf32. Our data suggests that Zcf32 regulates biofilm formation by repressing the expression of adhesins, chitinases and a significant number of other GPI-anchored proteins. We establish that there is the lesser recruitment of Zcf32 on the promoters of biofilm genes in biofilm condition compared to the planktonic mode of growth. Taking together, we propose that the transcription factor ZCF32 negatively regulates biofilm development in C. albicans. PMID:27498700

  14. Synthesis, structure and characterization of two new Zn(II) and Cd(II) complexes with the bidentate ligand 2-[(2-aminoethyl)amino]ethanaminium (AEEA)

    NASA Astrophysics Data System (ADS)

    Nbili, Wijdene; Zeller, Matthias; Lefebvre, Frédéric; Ben Nasr, Cherif

    2015-04-01

    Two new Zn(II) and Cd(II) complexes with the bidentate ligand 2-[(2-aminoethyl)amino]ethanaminium (AEEA), [ZnCl2(C4H14N3)]ClO4 (1) and [CdCl2(C4H14N3)]ClO4 (2), have been prepared and were characterized by single crystal X-ray diffraction, elemental analysis, CP-MAS-NMR and IR spectroscopies. The basic coordination patterns of the AEEA coordinated metal cations is highly distorted tetrahedral in (1) and distorted octahedral in (2). The crystal structure of (1) is characterized by ZnCl2N2 tetrahedra interconnected via N-H⋯Cl hydrogen bonds to form layers parallel to the (b, a + c) plane. In (2), the CdN2Cl4 entities form polymeric chains of edge-sharing octahedra extending along the a-axis direction. The crystal structures are stabilized by sets of hydrogen bonds, some of which are bi or trifurcated. The 13C and 15N CP-MAS NMR spectra are discussed and the vibrational absorption bands were identified by infrared spectroscopy.

  15. Theoretical investigations of the structures and electronic spectra of Zn(II) and Ni(II) complexes with cyclohexylamine-N-dithiocarbamate

    NASA Astrophysics Data System (ADS)

    Yu, Xiaohan; Wang, Na; He, Hongqing; Wang, Li

    2014-03-01

    The ground-state structures of two ligands cyclohexylamine-N-dithiocarbamate (L) and PPh3 and four complexes [Zn(L)2] (A), [Ni(L)2] (B), [Zn(L)2PPh3] (C), and [Ni(L)2PPh3] (D) are optimized by M06, B3LYP, and B3PW91 methods with the same mixed basis set. As compared with the experimental data of other complexes containing the Ni-P bond, the result obtained by M06/6-31+G(d)-LANL2DZ method is finally regarded as accurate and reliable for this project. Based on the optimized geometries, the compositions of molecular orbitals are analyzed and the absorption spectra are simulated. When one more ligand PPh3 is coordinated, the lowest-lying transition energy presents red-shift; while it shows blue-shift when the metal coordination center change from Ni to Zn with the same ligands. The detailed transition characters related with the absorption spectrum are assigned. In all the key transitions, it is hard to find the contribution from Zn atom. On the contrary, the d orbital of Ni atom contributes a lot for the HOMO and LUMO of complexes B and D. Consequently, the transition characters of Zn(II) and Ni(II) complexes are different.

  16. Synthesis, DFT Calculation, and Antimicrobial Studies of Novel Zn(II), Co(II), Cu(II), and Mn(II) Heteroleptic Complexes Containing Benzoylacetone and Dithiocarbamate

    PubMed Central

    Ekennia, Anthony C.; Onwudiwe, Damian C.; Olasunkanmi, Lukman O.; Osowole, Aderoju A.; Ebenso, Eno E.

    2015-01-01

    Heteroleptic complexes of zinc(II), copper(II), manganese(II), and cobalt(II) of the types [MLL′(H2O)2]·nH2O and [MLL′]·nH2O have been synthesized using sodium N-methyl-N-phenyldithiocarbamate (L) and benzoylacetone (L′). The metal complexes were characterized by elemental analysis, electrical conductance, magnetic susceptibility, infrared (IR), and UV-visible spectroscopic studies. The electrical conductance measurements revealed the nonelectrolytic nature of the synthesized complexes. The results of the elemental analyses, magnetic susceptibility measurements, and electronic spectra inferred that the Zn(II) complex adopted a four-coordinate geometry while the Co(II), Cu(II), and Mn(II) complexes assumed octahedral geometries. The IR spectra showed that the metal ions coordinated with the ligands via the S- and O-donor atoms. The geometry, electronic, and thermodynamic parameters of the complexes were obtained from density functional theory (DFT) calculations. The spin density distributions, relative strength of H–bonds, and thermodynamic parameters revealed that the order of stability of the metal complexes is Mn < Co < Cu > Zn. The agar diffusion methods were used to study the antimicrobial activity of the complexes against two Gram positive bacteria (S. aureus and S. pneumoniae), one Gram negative bacterium (E. coli), and two fungi organisms (A. niger and A. candida) and the complexes showed a broad spectrum of activities against the microbes. PMID:26681931

  17. Theoretical investigations of the structures and electronic spectra of Zn(II) and Ni(II) complexes with cyclohexylamine-N-dithiocarbamate.

    PubMed

    Yu, Xiaohan; Wang, Na; He, Hongqing; Wang, Li

    2014-03-25

    The ground-state structures of two ligands cyclohexylamine-N-dithiocarbamate (L) and PPh3 and four complexes [Zn(L)2] (A), [Ni(L)2] (B), [Zn(L)2PPh3] (C), and [Ni(L)2PPh3] (D) are optimized by M06, B3LYP, and B3PW91 methods with the same mixed basis set. As compared with the experimental data of other complexes containing the Ni-P bond, the result obtained by M06/6-31+G(d)-LANL2DZ method is finally regarded as accurate and reliable for this project. Based on the optimized geometries, the compositions of molecular orbitals are analyzed and the absorption spectra are simulated. When one more ligand PPh3 is coordinated, the lowest-lying transition energy presents red-shift; while it shows blue-shift when the metal coordination center change from Ni to Zn with the same ligands. The detailed transition characters related with the absorption spectrum are assigned. In all the key transitions, it is hard to find the contribution from Zn atom. On the contrary, the d orbital of Ni atom contributes a lot for the HOMO and LUMO of complexes B and D. Consequently, the transition characters of Zn(II) and Ni(II) complexes are different. PMID:24316543

  18. ZCF32, a fungus specific Zn(II)2 Cys6 transcription factor, is a repressor of the biofilm development in the human pathogen Candida albicans

    PubMed Central

    Kakade, Pallavi; Sadhale, Parag; Sanyal, Kaustuv; Nagaraja, Valakunja

    2016-01-01

    As a human fungal pathogen, Candida albicans can cause a wide variety of disease conditions ranging from superficial to systemic infections. Many of these infections are caused by an inherent ability of the pathogen to form biofilms on medical devices resulting in high mortality. Biofilms formed by C. albicans are a complex consortium of yeast and hyphal cells embedded in an extracellular matrix and are regulated by a network of transcription factors. Here, we report the role of a novel Zn(II)2-Cys6 binuclear cluster transcription factor, ZCF32, in the regulation of biofilm formation. Global transcriptome analysis reveals that biofilm development is the most altered pathway in the zcf32 null mutant. To delineate the functional correlation between ZCF32 and biofilm development, we determined the set of genes directly regulated by Zcf32. Our data suggests that Zcf32 regulates biofilm formation by repressing the expression of adhesins, chitinases and a significant number of other GPI-anchored proteins. We establish that there is the lesser recruitment of Zcf32 on the promoters of biofilm genes in biofilm condition compared to the planktonic mode of growth. Taking together, we propose that the transcription factor ZCF32 negatively regulates biofilm development in C. albicans. PMID:27498700

  19. Lasing the DNA fragments through β-diketimine framed Knoevenagel condensed Cu(II) and Zn(II) complexes - An in vitro and in vivo approach

    NASA Astrophysics Data System (ADS)

    Raman, Natarajan; Pravin, Narayanaperumal

    2014-01-01

    The syntheses, structures and spectroscopic properties of Cu(II) and Zn(II) complexes having Knoevenagel condensate β-diketimine Schiff base ligands have been investigated in this paper. Characterization of these complexes was carried out using FTIR, NMR, UV-Vis, elemental analysis, mass and EPR techniques. Absorption titration, electrochemical analyses and viscosity measurements have also been carried out to determine the mode of binding. The shift in ΔEp, E1/2 and Ipc values explores the interaction of CT DNA with the above metal complexes. Interaction of ligands and their complexes with DNA revealed an intercalative mode of binding between them. Antimicrobial studies showed an effective antimicrobial activity of the metal ions after coordination with the ligands. The antioxidant properties of the Schiff base ligands and their complexes were evaluated in a series of in vitro tests by using 1,1-diphenyl-2-picrylhydrazyl (DPPHrad ) and H2O2 free radical scavengers. In vivo and in vitro antitumor functions of the complexes against Ehrlich ascites carcinoma tumor model have also been investigated. All the results support that β-diketone derived Knoevenagel condensate Schiff base complexes may act as novel antitumor drugs and suggest that their potent cell life inhibition may contribute to their anti-cancer efficacy.

  20. Two new Zn(II) coordination polymers based on mixed pipemidic acid and flexible aromatic dicarboxylic acid ligands: Syntheses, crystal structures and luminescent properties

    NASA Astrophysics Data System (ADS)

    Jia, Yanxia; Zhou, Pingping

    2016-09-01

    Two new Zn(II) coordination polymers, namely [Zn(4,4‧-sdb) (HPPA)]n (1) and [Zn(2,2‧-bpdc)0.5(PPA)]n (2) (4,4‧-H2sdb = 4,4‧-sulfonyldibenzoate, 2,2‧-H2bpdc = 2,2‧-biphenyldicarboxylic acid, HPPA = pipemidic acid) were successfully obtained under hydrothermal conditions. These two compounds were further characterized by single-crystal X-ray diffraction analyses, elemental analyses, powder X-ray diffraction (PXRD) analyses and IR spectra. Compound 1 features a 1D chain structure, which further extended into a 3D supramolecular framework via intermolecular hydrogen bonds and weak van der Waals interactions, and compound 2 features a 3D framework with 6-connected α-Po-type topology. The structural regulation for these two compounds was successfully achieved by changing the flexible aromatic dicarboxylic acid ligand. Moreover, the thermal stabilities and luminescent properties for these two compounds were also investigated.

  1. Cu(II), Ni(II), and Zn(II) Complexes of Salan-Type Ligand Containing Ester Groups: Synthesis, Characterization, Electrochemical Properties, and In Vitro Biological Activities

    PubMed Central

    Jeslin Kanaga Inba, P.; Annaraj, B.; Thalamuthu, S.; Neelakantan, M. A.

    2013-01-01

    A salen ligand on reduction and N-alkylation affords a novel [N2O2] chelating ligand containing ester groups [L = diethyl-2,2′-(propane-1,3-diylbis((2-hydroxy-3-methoxy benzyl)azanediyl))diacetate]. The purity of the ligand was confirmed by NMR and HPLC chromatograms. Its Cu(II), Ni(II), and Zn(II) complexes were synthesized and characterized by a combination of elemental analysis, IR, NMR, UV-Vis, and mass spectral data, and thermogravimetric analysis (TG/DTA). The magnetic moments, UV-Vis, and EPR spectral studies support square planar geometry around the Cu(II) and Ni(II) ions. A tetrahedral geometry is observed in four-coordinate zinc with bulky N-alkylated salan ligand. The redox properties of the copper complex were examined in DMSO by cyclic voltammetry. The voltammograms show quasireversible process. The interaction of metal complexes with CT DNA was investigated by UV-Vis absorption titration, ethidium bromide displacement assay, cyclic voltammetry methods, and agarose gel electrophoresis. The apparent binding constant values suggest moderate intercalative binding modes between the complexes and DNA. The in vitro antioxidant and antimicrobial potentials of the synthesized compounds were also determined. PMID:23983672

  2. Tetra- and hexadentate Schiff base ligands and their Ni(II), Cu(II) and Zn(II) complexes. Synthesis, spectral, magnetic and thermal studies.

    PubMed

    Ismail, Tarek M A; Saleh, Akila A; El Ghamry, Mosad A

    2012-02-01

    Tetradentate N(2)O(2), N(4) Schiff bases, 1,2-bis(4-oxopent-2-ylideneamino) benzene (BOAB), 1-(4-oxopent-2-ylideneamino-2-[(2-hydroxyphenyl)ethylideneamino] benzene (OAHAB), 7,16-bis(4-chlorobenzylidene)-6,8,15,17-tetra-methyl-7,16-dihydro -5,9,14,18-tetraza-dibenzo[a,h] cyclo tetradecene (BCBDCT), 7,16-bis(2-hydroxy-benzylidene)-6,8,15,17-tetramethyl-7,16-dihydro-5,9,14,18-tetraza-dibenzo [a,h] cyclo tetradecene (BHBDCT) and hexadentate N(4)O(2) Schiff bases, 2,4-bis {2-[1-(2-hydroxyphenyl) ethylideneamino] phenylimino}-3-(2-hydroxybenzylidene) pentane (BHAPHP), 2,4-bis {2-[1-(2-hydroxyphenyl) ethylideneamino] phenylimino}-3-(4-chlorobenzylidene) pentane (BHAPCP) were prepared and characterized by elemental analysis, IR, UV-Vis, (1)H NMR and mass spectra. The solid complexes of the prepared Schiff base ligands with Ni(II), Cu(II) and Zn(II) ions were isolated and characterized by elemental and thermal analyses, IR, electronic and ESR spectra as well as conductance and magnetic susceptibility measurements. The results showed that most complexes have octahedral geometry but few can attain the tetrahedral arrangement. The TG analyses suggest high stability for most complexes followed by thermal decomposition in different steps. The kinetic and thermodynamic parameters for decomposition steps in Cu(II) complexes thermograms have been calculated. PMID:22070996

  3. Identification of InuR, a new Zn(II)2Cys6 transcriptional activator involved in the regulation of inulinolytic genes in Aspergillus niger.

    PubMed

    Yuan, Xiao-Lian; Roubos, Johannes A; van den Hondel, Cees A M J J; Ram, Arthur F J

    2008-01-01

    The expression of inulinolytic genes in Aspergillus niger is co-regulated and induced by inulin and sucrose. We have identified a positive acting transcription factor InuR, which is required for the induced expression of inulinolytic genes. InuR is a member of the fungal specific class of transcription factors of the Zn(II)2Cys6 type. Involvement of InuR in inulin and sucrose metabolism was suspected because of the clustering of inuR gene with sucB, which encodes an intracellular invertase with transfructosylation activity and a putative sugar transporter encoding gene (An15g00310). Deletion of the inuR gene resulted in a strain displaying a severe reduction in growth on inulin and sucrose medium. Northern analysis revealed that expression of inulinolytic and sucrolytic genes, e.g., inuE, inuA, sucA, as well as the putative sugar transporter gene (An15g00310) is dependent on InuR. Genome-wide expression analysis revealed, three additional putative sugar transporters encoding genes (An15g04060, An15g03940 and An17g01710), which were strongly induced by sucrose in an InuR dependent way. In silico analysis of the promoter sequences of strongly InuR regulated genes suggests that InuR might bind as dimer to two CGG triplets, which are separated by eight nucleotides. PMID:17917744

  4. Synthesis and characterization of an azo dibenzoic acid Schiff base and its Ni(II), Pb(II), Zn(II) and Cd(II) complexes

    NASA Astrophysics Data System (ADS)

    Kakanejadifard, Ali; Esna-ashari, Fatemeh; Hashemi, Payman; Zabardasti, Abedin

    2013-04-01

    The new Schiff base 4,4'-(1E,1'E)-(3,3'-(1E,1'E)-(pyridine-2,6-diylbis(azan-1-yl-1-ylid ene))bis(methan-1-yl-1-ylidene)bis(4-hydroxy-3,1-phenylene))bis(diazene-2,1-diyl)dibenzoic acid (1) was prepared from the condensation reaction of 2,6-diaminopyridine with 4-((3-formyl-4-hydroxyphenyl)diazenyl)benzoic acid in methanol. The compound 1 is potentially an N, O multidentate chelating ligand which could form stable complexes with metal ions in 1:1 up to 1:3 mol ratio of metal to ligand. The 1:1 complexes of Schiff base 1 with Ni(II), Pb(II), Zn(II) and Cd(II) have been synthesized by its condensation reaction with appropriate salts of metal ions. Structures of Schiff base (1) as well as its complexes with abovementioned metal ions were characterized by elemental analysis, mass, IR, UV-vis., 1H and 13С NMR spectroscopy.

  5. Tetra- and hexadentate Schiff base ligands and their Ni(II), Cu(II) and Zn(II) complexes. Synthesis, spectral, magnetic and thermal studies

    NASA Astrophysics Data System (ADS)

    Ismail, Tarek M. A.; Saleh, Akila A.; Ghamry, Mosad A. El

    2012-02-01

    Tetradentate N 2O 2, N 4 Schiff bases, 1,2-bis(4-oxopent-2-ylideneamino) benzene (BOAB), 1-(4-oxopent-2-ylideneamino-2-[(2-hydroxyphenyl)ethylideneamino] benzene (OAHAB), 7,16-bis(4-chlorobenzylidene)-6,8,15,17-tetra-methyl-7,16-dihydro -5,9,14,18-tetraza-dibenzo[a,h] cyclo tetradecene (BCBDCT), 7,16-bis(2-hydroxy-benzylidene)-6,8,15,17-tetramethyl-7,16-dihydro-5,9,14,18-tetraza-dibenzo [a,h] cyclo tetradecene (BHBDCT) and hexadentate N 4O 2 Schiff bases, 2,4-bis {2-[1-(2-hydroxyphenyl) ethylideneamino] phenylimino}-3-(2-hydroxybenzylidene) pentane (BHAPHP), 2,4-bis {2-[1-(2-hydroxyphenyl) ethylideneamino] phenylimino}-3-(4-chlorobenzylidene) pentane (BHAPCP) were prepared and characterized by elemental analysis, IR, UV-Vis, 1H NMR and mass spectra. The solid complexes of the prepared Schiff base ligands with Ni(II), Cu(II) and Zn(II) ions were isolated and characterized by elemental and thermal analyses, IR, electronic and ESR spectra as well as conductance and magnetic susceptibility measurements. The results showed that most complexes have octahedral geometry but few can attain the tetrahedral arrangement. The TG analyses suggest high stability for most complexes followed by thermal decomposition in different steps. The kinetic and thermodynamic parameters for decomposition steps in Cu(II) complexes thermograms have been calculated.

  6. Zn(II) and Cu(II) removal by Nostoc muscorum: a cyanobacterium isolated from a coal mining pit in Chiehruphi, Meghalaya, India.

    PubMed

    Goswami, Smita; Diengdoh, Omega L; Syiem, Mayashree B; Pakshirajan, Kannan; Kiran, Mothe Gopi

    2015-03-01

    Nostoc muscorum was isolated from a coal mining pit in Chiehruphi, Meghalaya, India, and its potential to remove Zn(II) and Cu(II) from media and the various biochemical alterations it undergoes during metal stress were studied. Metal uptake measured as a function of the ions removed by N. muscorum from media supplemented independently with 20 μmol/L ZnSO4 and CuSO4 established the ability of this cyanobacterium to remove 66% of Zn(2+) and 71% of Cu(2+) within 24 h of contact time. Metal binding on the cell surface was found to be the primary mode of uptake, followed by internalization. Within 7 days of contact, Zn(2+) and Cu(2+) mediated dissimilar effects on the organism. For instance, although chlorophyll a synthesis was increased by 12% in Zn(2+)-treated cells, it was reduced by 26% in Cu(2+)-treated cells. Total protein content remained unaltered in Zn(2+)-supplemented medium; however, a 15% reduction was noticed upon Cu(2+) exposure. Copper enhanced both photosynthesis and respiration by 15% and 19%, respectively; in contrast, photosynthesis was unchanged and respiration dropped by 11% upon Zn(2+) treatment. Inoculum age also influenced metal removal ability. Experiments in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (a photosynthetic inhibitor), carbonyl cyanide m-chlorophenyl hydrazone (an uncoupler), and exogenous ATP established that metal uptake was energy dependent, and photosynthesis contributed significantly towards the energy pool required to mediate metal removals. PMID:25670258

  7. Supramolecular complexes of Co(II), Ni(II) and Zn(II) p-hydroxybenzoates with caffeine: Synthesis, spectral characterization and crystal structure

    NASA Astrophysics Data System (ADS)

    Taşdemir, Erdal; Özbek, Füreya Elif; Sertçelik, Mustafa; Hökelek, Tuncer; Çelik, Raziye Çatak; Necefoğlu, Hacali

    2016-09-01

    Three novel complexes Co(II), Ni(II) and Zn(II) containing p-hydroxybenzoates and caffeine ligands were synthesized and characterized by elemental analysis, FT-IR and UV-vis Spectroscopy, molar conductivity and single crystal X-ray diffraction methods. The thermal properties of the synthesized complexes were investigated by TGA/DTA. The general formula of the complexes is [M(HOC6H4COO)2(H2O)4]·2(C8H10N4O2)·8H2O (where: M: Co, Ni and Zn). The IR studies showed that carboxylate groups of p-hydroxybenzoate ligands have monodentate coordination mode. The M2+ ions are octahedrally coordinated by two p-hydroxybenzoate ligands, four water molecules leading to an overall MO6 coordination environment. The medium-strength hydrogen bondings involving the uncoordinated caffeine ligands and water molecules, coordinated and uncoordinated water molecules and p-hydroxybenzoate ligands lead to three-dimensional supramolecular networks in the crystal structures.

  8. Lasing the DNA fragments through β-diketimine framed Knoevenagel condensed Cu(II) and Zn(II) complexes--an in vitro and in vivo approach.

    PubMed

    Raman, Natarajan; Pravin, Narayanaperumal

    2014-01-24

    The syntheses, structures and spectroscopic properties of Cu(II) and Zn(II) complexes having Knoevenagel condensate β-diketimine Schiff base ligands have been investigated in this paper. Characterization of these complexes was carried out using FTIR, NMR, UV-Vis, elemental analysis, mass and EPR techniques. Absorption titration, electrochemical analyses and viscosity measurements have also been carried out to determine the mode of binding. The shift in ΔEp, E1/2 and Ipc values explores the interaction of CT DNA with the above metal complexes. Interaction of ligands and their complexes with DNA revealed an intercalative mode of binding between them. Antimicrobial studies showed an effective antimicrobial activity of the metal ions after coordination with the ligands. The antioxidant properties of the Schiff base ligands and their complexes were evaluated in a series of in vitro tests by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and H2O2 free radical scavengers. In vivo and in vitro antitumor functions of the complexes against Ehrlich ascites carcinoma tumor model have also been investigated. All the results support that β-diketone derived Knoevenagel condensate Schiff base complexes may act as novel antitumor drugs and suggest that their potent cell life inhibition may contribute to their anti-cancer efficacy. PMID:24161850

  9. Development of a flow injection analysis (FIA) system for the measurement of heavy metals using a fiber optic chemical sensor based on laser-induced fluorescence

    NASA Astrophysics Data System (ADS)

    Zhang, Jingdong; Prestel, Harald; Gahr, Achim; Niessner, Reinhard

    2000-05-01

    The development of a fiber optic sensor system is described, for the on-line detection of heavy metal ions in water. This is based on laser-induced fluorescence spectroscopy of suitable metal-ligand complexes. The sensor system is designed to measure heavy metal ions in the field. Flow injection analysis (FIA) is coupled with the sensor system, to overcome problems of a slow diffusion rate of heavy metals through the membrane of an in situ sensor head. Preliminary experiments show the new FIA system has good reproducibility, a high sample analysis rate and it can measure heavy metal ions (Cu(II), Ni(II), Cd(II) and Zn(II)) at the ppb level, when using the appropriate ligands.

  10. Two Affinity Sites of the Cannabinoid Subtype 2 Receptor Identified by a Novel Homogeneous Binding Assay.

    PubMed

    Martínez-Pinilla, Eva; Rabal, Obdulia; Reyes-Resina, Irene; Zamarbide, Marta; Navarro, Gemma; Sánchez-Arias, Juan A; de Miguel, Irene; Lanciego, José L; Oyarzabal, Julen; Franco, Rafael

    2016-09-01

    Endocannabinoids act on G protein-coupled receptors that are considered potential targets for a variety of diseases. There are two different cannabinoid receptor types: ligands for cannabinoid type 2 receptors (CB2Rs) show more promise than those for cannabinoid type 1 receptors (CB1Rs) because they lack psychotropic actions. However, the complex pharmacology of these receptors, coupled with the lipophilic nature of ligands, is delaying the translational success of medications targeting the endocannabinoid system. We here report the discovery and synthesis of a fluorophore-conjugated CB2R-selective compound, CM-157 (3-[[4-[2-tert-butyl-1-(tetrahydropyran-4-ylmethyl)benzimidazol-5-yl]sulfonyl-2-pyridyl]oxy]propan-1-amine), which was useful for pharmacological characterization of CB2R by using a time-resolved fluorescence resonance energy transfer assay. This methodology does not require radiolabeled compounds and may be undertaken in homogeneous conditions and in living cells (i.e., without the need to isolate receptor-containing membranes). The affinity of the labeled compound was similar to that of the unlabeled molecule. Time-resolved fluorescence resonance energy transfer assays disclosed a previously unreported second affinity site and showed conformational changes in CB2R forming receptor heteromers with G protein-coupled receptor GPR55, a receptor for l-α-lysophosphatidylinositol. The populations displaying subnanomolar and nanomolar affinities were undisclosed in competitive assays using a well known cannabinoid receptor ligand, AM630 (1-[2-(morpholin-4-yl)ethyl]-2-methyl-3-(4-methoxybenzoyl)-6-iodoindole), and TH-chrysenediol, not previously tested on binding to cannabinoid receptors. Variations in binding parameters upon formation of dimers with GPR55 may reflect decreases in binding sites or alterations of the quaternary structure of the macromolecular G protein-coupled receptor complexes. In summary, the homogeneous binding assay described here may

  11. Analysis of conjugation of chloramphenicol and hemoglobin by fluorescence, circular dichroism and molecular modeling

    NASA Astrophysics Data System (ADS)

    Ding, Fei; Liu, Wei; Sun, Ye; Yang, Xin-Ling; Sun, Ying; Zhang, Li

    2012-01-01

    Chloramphenicol is a low cost, broad spectrum, highly active antibiotic, and widely used in the treatment of serious infections, including typhoid fever and other life-threatening infections of the central nervous system and respiratory tract. The purpose of the present study was to examine the conjugation of chloramphenicol with hemoglobin (Hb) and compared with albumin at molecular level, utilizing fluorescence, UV/vis absorption, circular dichroism (CD) as well as molecular modeling. Fluorescence data indicate that drug bind Hb generate quenching via static mechanism, this corroborates UV/vis absorption measurements that the ground state complex formation with an affinity of 10 4 M -1, and the driving forces in the Hb-drug complex are hydrophilic interactions and hydrogen bonds, as derived from computational model. The accurate binding site of drug has been identified from the analysis of fluorescence and molecular modeling, α1β2 interface of Hb was assigned to possess high-affinity for drug, which located at the β-37 Trp nearby. The structural investigation of the complexed Hb by synchronous fluorescence, UV/vis absorption, and CD observations revealed some degree of Hb structure unfolding upon complexation. Based on molecular modeling, we can draw the conclusion that the binding affinity of drug with albumin is superior, compared with Hb. These phenomena can provide salient information on the absorption, distribution, pharmacology, and toxicity of chloramphenicol and other drugs which have analogous configuration with chloramphenicol.

  12. Inducible fluorescent speckle microscopy

    PubMed Central

    Aguiar, Paulo; Belsley, Michael; Maiato, Helder

    2016-01-01

    The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration. PMID:26783303

  13. Fluorescence and Light Scattering

    ERIC Educational Resources Information Center

    Clarke, Ronald J.; Oprysa, Anna

    2004-01-01

    The aim of the mentioned experiment is to aid students in developing tactics for distinguishing between signals originating from fluorescence and light scattering. Also, the experiment provides students with a deeper understanding of the physicochemical bases of each phenomenon and shows that the techniques are actually related.

  14. Ultraviolet fluorescence monitor

    SciTech Connect

    Hargis, P.J. Jr.; Preppernau, B.L.; Aragon, B.P.

    1997-05-01

    A multispectral ultraviolet (UV) fluorescence imaging fluorometer and a pulsed molecular beam laser fluorometer were developed to detect volatile organic compounds of interest in environmental monitoring and drug interdiction applications. The UV fluorescence imaging fluorometer is a relatively simple instrument which uses multiple excitation wavelengths to measure the excitation/emission matrix for irradiated samples. Detection limits in the high part-per-million to low part-per-million range were measured for a number of volatile organic vapors in the atmosphere. Detection limits in the low part-per-million range were obtained using cryogenic cooling to pre-concentrate unknown samples before introducing them into the imaging fluorometer. A multivariate analysis algorithm was developed to analyze the excitation/emission matrix and used to determine the relative concentrations of species in computer synthesized mixtures containing up to five organic compounds. Analysis results demonstrated the utility of multispectral UV fluorescence in analytical measurements. A transportable UV fluorescence imaging fluorometer was used in two field tests. Field test results demonstrated that detection limits in the part-per-billion range were needed to reliably identify volatile organic compounds in realistic field test measurements. The molecular beam laser fluorometer, a more complex instrument with detection limits in the part-per-billion to part-per-trillion range, was therefore developed to satisfy detection sensitivity requirements for field test measurements. High-resolution spectroscopic measurements made with the molecular beam laser fluorometer demonstrated its utility in identifying volatile organic compounds in the atmosphere.

  15. Inducible fluorescent speckle microscopy.

    PubMed

    Pereira, António J; Aguiar, Paulo; Belsley, Michael; Maiato, Helder

    2016-01-18

    The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration. PMID:26783303

  16. Fluorescent Gage Indication

    NASA Technical Reports Server (NTRS)

    Barns, C. E.; Gilbaugh, B. L.; Gin, B.; Holt, W. L.; Lesak, P.; Mancini, R.; Spencer, H. F.

    1985-01-01

    Transfer of dye shows quality of contact between two mating parts. Mating parts checked for fit by spreading fluorescent dye on one, making brief light contact with other, and looking (under UV light) for transferred dye. Dye offers greater visibility under ultraviolet illumination, allowing better indication of how precisely parts match and what areas interfere.

  17. Fluorescence Imaging in Surgery

    PubMed Central

    Orosco, Ryan K.; Tsien, Roger Y.; Nguyen, Quyen T.

    2013-01-01

    Although the modern surgical era is highlighted by multiple technological advances and innovations, one area that has remained constant is the dependence of the surgeon's vision on white-light reflectance. This renders different body tissues in a limited palette of various shades of pink and red, thereby limiting the visual contrast available to the operating surgeon. Healthy tissue, anatomic variations, and diseased states are seen as slight discolorations relative to each other and differences are inherently limited in dynamic range. In the upcoming years, surgery will undergo a paradigm shift with the use of targeted fluorescence imaging probes aimed at augmenting the surgical armamentarium by expanding the “visible” spectrum available to surgeons. Such fluorescent “smart probes” will provide real-time, intraoperative, pseudo-color, high-contrast delineation of both normal and pathologic tissues. Fluorescent surgical molecular guidance promises another major leap forward to improve patient safety and clinical outcomes, and to reduce overall healthcare costs. This review provides an overview of current and future surgical applications of fluorescence imaging in diseased and nondiseased tissues and focus on the innovative fields of image processing and instrumentation. PMID:23335674

  18. Detecting and Quantifying Biomolecular Interactions of a Dendritic Polyglycerol Sulfate Nanoparticle Using Fluorescence Lifetime Measurements.

    PubMed

    Boreham, Alexander; Pikkemaat, Jens; Volz, Pierre; Brodwolf, Robert; Kuehne, Christian; Licha, Kai; Haag, Rainer; Dernedde, Jens; Alexiev, Ulrike

    2015-01-01

    Interactions of nanoparticles with biomaterials determine the biological activity that is key for the physiological response. Dendritic polyglycerol sulfates (dPGS) were found recently to act as an inhibitor of inflammation by blocking selectins. Systemic application of dPGS would present this nanoparticle to various biological molecules that rapidly adsorb to the nanoparticle surface or lead to adsorption of the nanoparticle to cellular structures such as lipid membranes. In the past, fluorescence lifetime measurements of fluorescently tagged nanoparticles at a molecular and cellular/tissue level have been proven to reveal valuable information on the local nanoparticle environment via characteristic fluorescent lifetime signatures of the nanoparticle bound dye. Here, we established fluorescence lifetime measurements as a tool to determine the binding affinity to fluorescently tagged dPGS (dPGS-ICC; ICC: indocarbocyanine). The binding to a cell adhesion molecule (L-selectin) and a human complement protein (C1q) to dPGS-ICC was evaluated by the concentration dependent change in the unique fluorescence lifetime signature of dPGS-ICC. The apparent binding affinity was found to be in the nanomolar range for both proteins (L-selectin: 87 ± 4 nM and C1q: 42 ± 12 nM). Furthermore, the effect of human serum on the unique fluorescence lifetime signature of dPGS-ICC was measured and found to be different from the interactions with the two proteins and lipid membranes. A comparison between the unique lifetime signatures of dPGS-ICC in different biological environments shows that fluorescence lifetime measurements of unique dPGS-ICC fluorescence lifetime signatures are a versatile tool to probe the microenvironment of dPGS in cells and tissue. PMID:26712722

  19. Bimolecular fluorescence complementation.

    PubMed

    Wong, Katy A; O'Bryan, John P

    2011-01-01

    Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1). A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and Förster resonance energy transfer (FRET). For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET

  20. Binding Affinity Effects on Physical Characteristics of a Model Phase-Separated Protein Droplet

    NASA Astrophysics Data System (ADS)

    Chuang, Sara; Banani, Salman; Rosen, Michael; Brangwynne, Clifford

    2015-03-01

    Non-membrane bound organelles are associated with a range of biological functions. Several of these structures exhibit liquid-like properties, and may represent droplets of phase-separated RNA and/or proteins. These structures are often enriched in multi-valent molecules, however little is known about the interactions driving the assembly, properties, and function. Here, we address this question using a model multi-valent protein system consisting of repeats of Small Ubiquitin-like Modifier (SUMO) protein and a SUMO-interacting motif (SIM). These proteins undergo phase separation into liquid-like droplets. We combine microrheology and quantitative microscopy to determine affect of binding affinity on the viscosity, density and surface tension of these droplets. We also use fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and partitioning experiments to probe the structure and dynamics within these droplets. Our results shed light on how inter-molecular interactions manifests in droplet properties, and lay the groundwork for a comprehensive biophysical picture of intracellular RNA/protein organelles.

  1. Green fluorescence induced by EF-hand assembly in a split GFP system

    PubMed Central

    Lindman, Stina; Johansson, Ida; Thulin, Eva; Linse, Sara

    2009-01-01

    The affinity between the 1–157 and 158–238 fragments of green fluorescent protein (GFP) is too low for spontaneous in vivo reassembly of the protein upon co-expression of the two fragments. This prevents chromophore maturation and the cells lack GFP fluorescence. We have utilized the very high affinity between the two EF-hands of calbindin D9k to facilitate GFP assembly from its fragments and to introduce a calcium dependent molecular switch. In GFPN-EF1, residues 1–157 of GFP are fused to residues 1–43 of calbindin, and in EF2-GFPC, residues 44–75 of calbindin are fused to residues 158–238 of GFP. When co-expressed, GFPN-EF1 and EF2-GFPC associate spontaneously and rapidly resulting in a folded reconstituted protein with bright GFP fluorescence. The high affinity of GFPN-EF1 for EF2-GFPC leads to brighter fluorescence of the cells compared to cells with a control constructs carrying leucine zippers (Wilson et al., Nature Methods 2004;3:255). The complex of GFPN-EF1 and EF2-GFPC was purified from cells using metal-ion chelate chromatography and the temperature dependence of GFP fluorescence was found to be calcium dependent. The GFPN-EF1 and EF2-GFPC fragments were separated by ion exchange chromatography. The assembly of the fragments was found to be reversible and the complex was regained upon mixing, as evidenced by surface plasmon resonance (SPR) data. The affinity between GFPN-EF1 and EF2-GFPC as well as rates of association and dissociation were found to be Ca2+-dependent. PMID:19472338

  2. Green fluorescence induced by EF-hand assembly in a split GFP system.

    PubMed

    Lindman, Stina; Johansson, Ida; Thulin, Eva; Linse, Sara

    2009-06-01

    The affinity between the 1-157 and 158-238 fragments of green fluorescent protein (GFP) is too low for spontaneous in vivo reassembly of the protein upon co-expression of the two fragments. This prevents chromophore maturation and the cells lack GFP fluorescence. We have utilized the very high affinity between the two EF-hands of calbindin D(9k) to facilitate GFP assembly from its fragments and to introduce a calcium dependent molecular switch. In GFPN-EF1, residues 1-157 of GFP are fused to residues 1-43 of calbindin, and in EF2-GFPC, residues 44-75 of calbindin are fused to residues 158-238 of GFP. When co-expressed, GFPN-EF1 and EF2-GFPC associate spontaneously and rapidly resulting in a folded reconstituted protein with bright GFP fluorescence. The high affinity of GFPN-EF1 for EF2-GFPC leads to brighter fluorescence of the cells compared to cells with a control constructs carrying leucine zippers (Wilson et al., Nature Methods 2004;3:255). The complex of GFPN-EF1 and EF2-GFPC was purified from cells using metal-ion chelate chromatography and the temperature dependence of GFP fluorescence was found to be calcium dependent. The GFPN-EF1 and EF2-GFPC fragments were separated by ion exchange chromatography. The assembly of the fragments was found to be reversible and the complex was regained upon mixing, as evidenced by surface plasmon resonance (SPR) data. The affinity between GFPN-EF1 and EF2-GFPC as well as rates of association and dissociation were found to be Ca(2+)-dependent. PMID:19472338

  3. Excited Protein States of Human Tear Lipocalin for Low- and High-Affinity Ligand Binding Revealed by Functional AB Loop Motion

    PubMed Central

    Gasymov, Oktay K.; Abduragimov, Adil R.; Glasgow, Ben J.

    2010-01-01

    Human tear lipocalin (TL), a prominent member of lipocalin family, exhibits functional and structural promiscuity. The plasticity of loop regions modulates entry to the ligand pocket at the “open” end of the eight-stranded β-barrel. Site directed multi-distance measurements using fluorescence resonance energy transfer between functional loops register two excited protein states for low- and high-affinity ligand binding. At low pH, the longest loop AB adopts the conformation of the low-affinity excited protein state that matches the crystal structure of holo-TL at pH 8. A “crankshaft” like movement is detected for the loop AB in a low pH transition. At pH 7.3 the holo-protein assumes a high-affinity excited protein state, in which the loop AB is more compact (RMS= 3.1Å). In the apo-holo transition, the reporter Trp 28 moves about 4.5 Å that reflects a decrease in distance between Glu27 and Lys108. This interaction fixes the loop AB conformation for the high-affinity mode. No such of movement is detected at low pH, where Glu27 is protonated. Data strongly indicate that the protonation state of Glu27 modulates the conformation of the loop AB for high- and low-affinity binding. PMID:20439130

  4. Excited protein states of human tear lipocalin for low- and high-affinity ligand binding revealed by functional AB loop motion.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2010-06-01

    Human tear lipocalin (TL), a prominent member of lipocalin family, exhibits functional and structural promiscuity. The plasticity of loop regions modulates entry to the ligand pocket at the "open" end of the eight-stranded beta-barrel. Site-directed multi-distance measurements using fluorescence resonance energy transfer between functional loops register two excited protein states for low- and high-affinity ligand binding. At low pH, the longest loop AB adopts the conformation of the low-affinity excited protein state that matches the crystal structure of holo-TL at pH 8. A "crankshaft" like movement is detected for the loop AB in a low pH transition. At pH 7.3 the holo-protein assumes a high-affinity excited protein state, in which the loop AB is more compact (RMS=3.1A). In the apo-holo transition, the reporter Trp 28 moves about 4.5A that reflects a decrease in distance between Glu27 and Lys108. This interaction fixes the loop AB conformation for the high-affinity mode. No such movement is detected at low pH, where Glu27 is protonated. Data strongly indicate that the protonation state of Glu27 modulates the conformation of the loop AB for high- and low-affinity binding. PMID:20439130

  5. Fluorescence Microscopy of Single Molecules

    ERIC Educational Resources Information Center

    Zimmermann, Jan; van Dorp, Arthur; Renn, Alois

    2004-01-01

    The investigation of photochemistry and photophysics of individual quantum systems is described with the help of a wide-field fluorescence microscopy approach. The fluorescence single molecules are observed in real time.

  6. A nitrogen-dependent switch in the high affinity ammonium transport in Medicago truncatula.

    PubMed

    Straub, Daniel; Ludewig, Uwe; Neuhäuser, Benjamin

    2014-11-01

    Ammonium transporters (AMTs) are crucial for the high affinity primary uptake and translocation of ammonium in plants. In the model legume Medicago truncatula, the genomic set of AMT-type ammonium transporters comprises eight members. Only four genes were abundantly expressed in young seedlings, both in roots and shoots. While the expression of all AMTs in the shoot was not affected by the nitrogen availability, the dominating MtAMT1;1 gene was repressed by nitrogen in roots, despite that cellular nitrogen concentrations were far above deficiency levels. A contrasting de-repression by nitrogen was observed for MtAMT1;4 and MtAMT2;1, which were both expressed at intermediate level. Weak expression was found for MtAMT1;2 and MtAMT2;3, while the other AMTs were not detected in young seedlings. When expressed from their endogenous promoters, translational fusion proteins of MtAMT1;1 and MtAMT2;1 with green fluorescent protein were co-localized in the plasma membrane of rhizodermal cells, but also detected in cortical root layers. Both transporter proteins similarly functionally complemented a yeast strain that is deficient in high affinity ammonium transport, both at acidic and neutral pH. The uptake into yeast mediated by these transporters saturated with Km AMT1;1 = 89 µM and Km AMT2;1 = 123 µM, respectively. When expressed in oocytes, MtAMT1;1 mediated much larger (15)N-ammonium uptake than MtAMT2;1, but NH4 (+) currents were only recorded for MtAMT1;1. These currents saturated with a voltage-dependent Km = 90 µM at -80 mV. The cellular localization and regulation of the AMTs suggests that MtAMT1;1 encodes the major high affinity ammonium transporter gene in low nitrogen grown young M. truncatula roots and despite the similar localization and substrate affinity, MtAMT2;1 appears functionally distinct and more important at higher nitrogen supply. PMID:25164101

  7. A universal approach to ultrasmall magneto-fluorescent nanohybrids.

    PubMed

    Feld, Artur; Merkl, Jan-Philip; Kloust, Hauke; Flessau, Sandra; Schmidtke, Christian; Wolter, Christopher; Ostermann, Johannes; Kampferbeck, Michael; Eggers, Robin; Mews, Alf; Schotten, Theo; Weller, Horst

    2015-10-12

    Seeded emulsion polymerization is a powerful universal method to produce ultrasmall multifunctional magnetic nanohybrids. In a two-step procedure, iron oxide nanocrystals were initially encapsulated in a polystyrene (PS) shell and subsequently used as beads for a controlled assembly of elongated quantum dots/quantum rods (QDQRs). The synthesis of a continuous PS shell allows the whole construct to be fixed and the composition of the nanohybrid to be tuned. The fluorescence of the QDQRs and magnetism of iron oxide were perfectly preserved, as confirmed by single-particle investigation, fluorescence decay measurements, and relaxometry. Bio-functionalization of the hybrids was straightforward, involving copolymerization of appropriate affinity ligands as shown by immunoblot analysis. Additionally, the universality of this method was shown by the embedment of a broad scale of NPs. PMID:26136318

  8. Increased hemoglobin O2 affinity protects during acute hypoxia.

    PubMed

    Yalcin, Ozlem; Cabrales, Pedro

    2012-08-01

    Acclimatization to hypoxia requires time to complete the adaptation mechanisms that influence oxygen (O(2)) transport and O(2) utilization. Although decreasing hemoglobin (Hb) O(2) affinity would favor the release of O(2) to the tissues, increasing Hb O(2) affinity would augment arterial O(2) saturation during hypoxia. This study was designed to test the hypothesis that pharmacologically increasing the Hb O(2) affinity will augment O(2) transport during severe hypoxia (10 and 5% inspired O(2)) compared with normal Hb O(2) affinity. RBC Hb O(2) affinity was increased by infusion of 20 mg/kg of 5-hydroxymethyl-2-furfural (5HMF). Control animals received only the vehicle. The effects of increasing Hb O(2) affinity were studied in the hamster window chamber model, in terms of systemic and microvascular hemodynamics and partial pressures of O(2) (Po(2)). Pimonidazole binding to hypoxic areas of mice heart and brain was also studied. 5HMF decreased the Po(2) at which the Hb is 50% saturated with O(2) by 12.6 mmHg. During 10 and 5% O(2) hypoxia, 5HMF increased arterial blood O(2) saturation by 35 and 48% from the vehicle group, respectively. During 5% O(2) hypoxia, blood pressure and heart rate were 58 and 30% higher for 5HMF compared with the vehicle. In addition, 5HMF preserved microvascular blood flow, whereas blood flow decreased to 40% of baseline in the vehicle group. Consequently, perivascular Po(2) was three times higher in the 5HMF group compared with the control group at 5% O(2) hypoxia. 5HMF also reduced heart and brain hypoxic areas in mice. Therefore, increased Hb O(2) affinity resulted in hemodynamics and oxygenation benefits during severe hypoxia. This acute acclimatization process may have implications in survival during severe environmental hypoxia when logistic constraints prevent chronic acclimatization. PMID:22636677

  9. Affinities of methylphenidate derivatives for dopamine, norepinephrine and serotonin transporters.

    PubMed

    Gatley, S J; Pan, D; Chen, R; Chaturvedi, G; Ding, Y S

    1996-01-01

    We have synthesized several derivative of dl-threo-methylphenidate (Ritalin) bearing substituents on the phenyl ring. IC50 values for binding these compounds to rat brain monoamine transporters were assessed using [3H]WIN 35,428 (striatal membranes, dopamine transporters, DAT), [3H]nisoxetine (frontal cortex membranes, norepinephrine transporters, NET) and [3H]paroxetine (brain stem membranes, 5HT transporters, 5HTT). Affinities (1/Ki) decreased in the order: DAT > NET > 5HTT. Substitution at the para position of dl-threo-methylphenidate generally led to retained or increased affinity for the dopamine transporter (bromo > iodo > methoxy > hydroxy). Substitution at the meta position also increased affinity for the DAT (m-bromo > methylphenidate; m-iodo-p-hydroxy > p-hydroxy). Substitution at the ortho position with bromine considerably decreased affinity. Similar IC50 values for binding of o-bromomethylphenidate to the dopamine transporter were measured at 0, 22 and 37 degrees. N-Methylation of the piperidine ring of methylphenidate also considerably reduced affinity. The dl-erythro isomer of o-bromomethylphenidate did not bind to the DAT (IC50 > 50,000 nM). Affinities at the dopamine and norepinephrine transporters for substituted methylphenidate derivatives were well correlated (r2=0.90). Abilities of several methylphenidate derivatives to inhibit [3H]dopamine uptake in striatal synaptosomes corresponded well with inhibition of [3H]WIN 35, 428 binding. None of the compounds examined exhibited significant affinity to dopamine D1 or D2 receptors (IC50 > 500 or 5,000 nM, respectively), as assessed by inhibition of binding of [3H]SCH 23390 or [123I]epidepride, respectively, to striatal membranes. PMID:8786705

  10. Increased hemoglobin O2 affinity protects during acute hypoxia

    PubMed Central

    Yalcin, Ozlem

    2012-01-01

    Acclimatization to hypoxia requires time to complete the adaptation mechanisms that influence oxygen (O2) transport and O2 utilization. Although decreasing hemoglobin (Hb) O2 affinity would favor the release of O2 to the tissues, increasing Hb O2 affinity would augment arterial O2 saturation during hypoxia. This study was designed to test the hypothesis that pharmacologically increasing the Hb O2 affinity will augment O2 transport during severe hypoxia (10 and 5% inspired O2) compared with normal Hb O2 affinity. RBC Hb O2 affinity was increased by infusion of 20 mg/kg of 5-hydroxymethyl-2-furfural (5HMF). Control animals received only the vehicle. The effects of increasing Hb O2 affinity were studied in the hamster window chamber model, in terms of systemic and microvascular hemodynamics and partial pressures of O2 (Po2). Pimonidazole binding to hypoxic areas of mice heart and brain was also studied. 5HMF decreased the Po2 at which the Hb is 50% saturated with O2 by 12.6 mmHg. During 10 and 5% O2 hypoxia, 5HMF increased arterial blood O2 saturation by 35 and 48% from the vehicle group, respectively. During 5% O2 hypoxia, blood pressure and heart rate were 58 and 30% higher for 5HMF compared with the vehicle. In addition, 5HMF preserved microvascular blood flow, whereas blood flow decreased to 40% of baseline in the vehicle group. Consequently, perivascular Po2 was three times higher in the 5HMF group compared with the control group at 5% O2 hypoxia. 5HMF also reduced heart and brain hypoxic areas in mice. Therefore, increased Hb O2 affinity resulted in hemodynamics and oxygenation benefits during severe hypoxia. This acute acclimatization process may have implications in survival during severe environmental hypoxia when logistic constraints prevent chronic acclimatization. PMID:22636677

  11. Fluorescence lifetime analysis and effect of magnesium ions on binding of NADH to human aldehyde dehydrogenase 1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aldehyde dehydrogenase 1 (ALDH1) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence ALDH1 activity in part by increasing NADH binding affinity to the enzyme thus reducing activity. By using time-resolved fluorescence spectroscopy, we have resolved t...

  12. Measuring Norfloxacin Binding to Trypsin Using a Fluorescence Quenching Assay in an Upper-Division, Integrated Laboratory Course

    ERIC Educational Resources Information Center

    Hicks, Katherine A.

    2016-01-01

    Fluorescence quenching assays are often used to measure dissociation constants that quantify the binding affinity between small molecules and proteins. In an upper-division undergraduate laboratory course, where students work on projects using a guided inquiry-based approach, a binding titration experiment at physiological pH is performed to…

  13. A spiropyran-based fluorescent probe for the specific detection of β-amyloid peptide oligomers in Alzheimer's disease.

    PubMed

    Lv, Guanglei; Sun, Anyang; Wei, Peng; Zhang, Ning; Lan, Haichuang; Yi, Tao

    2016-07-01

    We report a new spiropyran-based fluorescent probe that exhibits high affinity and specificity towards Aβ oligomers both in vitro and in vivo. This probe can penetrate the blood brain barrier and specifically target Aβ oligomers in the brains of transgenic mice in models for Alzheimer's disease. PMID:27346489

  14. Dansyl labeling to modulate the relative affinity of bile acids for the binding sites of human serum albumin.

    PubMed

    Rohacova, Jana; Sastre, German; Marin, M Luisa; Miranda, Miguel A

    2011-09-01

    Binding of natural bile acids to human serum albumin (HSA) is an important step in enterohepatic circulation and provides a measure of liver function. In this article, we report on the use of four dansyl (Dns) derivatives of cholic acid (ChA) to demonstrate a regiodifferentiation in their relative affinity for the two binding sites of HSA. Using both steady-state and time-resolved fluorescence, formation of Dns-ChA@HSA complexes was confirmed; the corresponding binding constants were determined, and their distribution between bulk solution and HSA microenvironment was estimated. By means of energy transfer from Trp to the Dns moiety, donor-acceptor distances were estimated (21-25 Å) and found to be compatible with both site 1 and site 2 occupancies. Nevertheless, titration using warfarin and ibuprofen as specific displacement probes clearly indicated that 3α- and 3β-Dns-ChA bind to HSA at site 2, whereas their C-7 regioisomers bind to HSA at site 1. Furthermore, the C-3-labeled compounds are displaced by lithocholic acid, whereas they are insensitive to ChA, confirming the assumption that the former binds to HSA at site 2. Thus, Dns labeling provides a useful tool to modulate the relative affinity of ChA to the major binding sites of HSA and, in combination with other fluorescent ChA analogs, to mimic the binding behavior of natural bile acids. PMID:21797258

  15. Affinities of organophosphate flame retardants to tumor suppressor gene p53: an integrated in vitro and in silico study.

    PubMed

    Li, Fei; Cao, Lulu; Li, Xuehua; Li, Na; Wang, Zijian; Wu, Huifeng

    2015-01-22

    Health concerns have been raised in regards to the environmental impact of the more frequently used organophosphate flame retardants (OPFRs). In this study, the effects of two typical OPFRs (TCPP and TPhP) on p53 gene expression in human embryo liver L02 cells were determined by quantitative real-time PCR. To better understand the relationship between molecular structural features of OPFRs and binding affinities for the tumor suppressor genes p53, an integrated experimental and in silico approach was used. The interaction of 9 OPFRs with p53 DNA fragment under simulated physiological conditions (phosphate buffer solution of pH 7.40), was explored by UV absorption spectroscopy, fluorescence spectroscopy and molecular modeling method. The binding constants of 9 OPFRs with p53 DNA fragment were determined respectively, using ethidium bromide (EB) as fluorescence probe of DNA. From docking analysis, hydrogen bonding and hydrophobic interactions were found to be the dominant interactions. Based on the observed interactions, appropriate molecular structural parameters were adopted to develop a quantitative structure-activity relationship (QSAR) model. The binding affinities of OPFRs to p53 DNA fragment were related with molecular electrostatic potential. The developed QSAR model had good robustness, predictive ability and mechanism interpretability. PMID:25510514

  16. Fluorescent noble metal nanoclusters

    NASA Astrophysics Data System (ADS)

    Zheng, Jie

    Water-soluble fluorescent metallic clusters at sizes comparable to the Fermi wavelength of an electron (˜0.5 nm for gold and silver) were created and their photophysical properties were investigated at the bulk and single molecule levels. We employed biocompatible dendrimer and peptide to prepare a series of strong fluorescent gold and silver clusters with chemical or photo reduction methods. Facilitated by the well-defined dendrimer size, electrospray ionization mass spectrometry indicates that the fluorescent silver nanocluster size ranges from 2 to 8 Ag atoms. The correlation of emission energy with the number of atoms, N, in each gold nanocluster is quantitatively fit for the smallest nanoclusters with no adjustable parameters by the simple scaling relation of EFermi/N1/3, in which EFermi is the Fermi energy of bulk gold. The transition energy scaling inversely with cluster radius indicates that electronic structure can be well described with the spherical jellium model and further demonstrates that these nanomaterials are "multi-electron artificial atoms". Fluorescence from these small metal clusters can be considered protoplasmonic, molecular transitions of the free conduction electrons before the onset of collective dipole oscillations occurring when a continuous density of states is reached. In addition, very strong single molecular Stokes and anti-Stokes Raman enhancement by fluorescent silver clusters was observed. Pushing to larger sizes, we also created ˜2nm diameter glutathione encapsulated luminescent gold nanoparticles. Distinct from similarly sized but nonluminescent gold nanoparticles, these 2 nm gold nanoparticles show bright, long lifetime emission but no plasmon absorption. The emission might arise from charge transfer between gold atoms and the thiol ligand. Providing the "missing link" between atomic and nanoparticle behavior in noble metals, these highly fluorescent, water-soluble gold and silver nanoclusters offer complementary transition

  17. Crystal structure and ligand affinity of avidin in the complex with 4‧-hydroxyazobenzene-2-carboxylic acid

    NASA Astrophysics Data System (ADS)

    Strzelczyk, Paweł; Bujacz, Grzegorz

    2016-04-01

    Avidin is a protein found in egg white that binds numerous organic compounds with high affinity, especially biotin and its derivatives. Due to its extraordinary affinity for its ligands, avidin is extensively used in biotechnology. X-ray crystallography and fluorescence-based biophysical techniques were used to show that avidin binds the dye 4‧-hydroxyazobenzene-2-carboxylic acid (HABA) with a lower affinity than biotin. The apparent dissociation constant determined for the avidin complex with HABA by microscale thermophoresis (MST) is 4.12 μM. The crystal structure of avidin-HABA complex was determined at a resolution of 2.2 Å (PDB entry 5chk). The crystals belong to a hexagonal system, in the space group P6422. In that structure, the hydrazone tautomer of HABA is bound at the bottom part of the central calyx near the polar residues. We show interactions of the dye with avidin and compare them with the previously reported avidin-biotin complex.

  18. Low-affinity transport of FITC-albumin in alveolar type II epithelial cell line RLE-6TN.

    PubMed

    Tagawa, Maki; Yumoto, Ryoko; Oda, Keisuke; Nagai, Junya; Takano, Mikihisa

    2008-01-01

    FITC-albumin uptake by cultured alveolar type II epithelial cells, RLE-6TN, is mediated by high- and low-affinity transport systems. In this study, characteristics of the low-affinity transport system were evaluated. The uptake of FITC-albumin was time and temperature dependent and was inhibited by metabolic inhibitors and bafilomycin A1. Confocal laser scanning microscopic analysis showed punctate localization of the fluorescence in the cells, which was partly localized in lysosomes. FITC-albumin taken up by the cells gradually degraded over time, as shown by fluoroimage analyzer after SDS-PAGE. The uptake of FITC-albumin by RLE-6TN cells was not inhibited by caveolae-mediated endocytosis inhibitors such as nystatin, but was inhibited by clathrin-mediated endocytosis inhibitors such as phenylarsine oxide. The uptake was also inhibited by potassium depletion and hypertonicity, conditions known to inhibit clathrin-mediated endocytosis. In addition, macropinocytosis inhibitors such as 5-(N-ethyl-N-isopropyl) amiloride inhibited the uptake. These results indicate that the low-affinity transport of FITC-albumin in RLE-6TN cells is at least in part mediated by clathrin-mediated endocytosis, but not by caveolae-mediated endocytosis. Possible involvement of macropinocytosis was also suggested. PMID:18974609

  19. Crystal structure and ligand affinity of avidin in the complex with 4‧-hydroxyazobenzene-2-carboxylic acid

    NASA Astrophysics Data System (ADS)

    Strzelczyk, Paweł; Bujacz, Grzegorz

    2016-04-01

    Avidin is a protein found in egg white that binds numerous organic compounds with high affinity, especially biotin and its derivatives. Due to its extraordinary affinity for its ligands, avidin is extensively used in biotechnology. X-ray crystallography and fluorescence-based biophysical techniques were used to show that avidin binds the dye 4‧-hydroxyazobenzene-2-carboxylic acid (HABA) with a lower affinity than biotin. The apparent dissociation constant determined for the avidin complex with HABA by microscale thermophoresis (MST) is 4.12 μM. The crystal structure of avidin-HABA complex was determined at a resolution of 2.2 Å (PDB entry 5chk). The crystals belong to a hexagonal system, in the space group P6422. In that structure, the hydrazone tautomer of HABA is bound at the bottom part of the central calyx near the polar residues. We show interactions of the dye with avidin and compare them with the previously reported avidin-biotin complex.

  20. Robust Affinity Standards for Cu(I) Biochemistry

    PubMed Central

    Bagchi, Pritha; Morgan, M. Thomas; Bacsa, John; Fahrni, Christoph J.

    2014-01-01

    The measurement of reliable Cu(I) protein binding affinities requires competing reference ligands with similar binding strengths; however, the literature on such reference ligands is not only sparse but often conflicting. To address this deficiency, we have created and characterized a series of water-soluble monovalent copper ligands, MCL-1, MCL-2, and MCL-3, that form well-defined, air-stable, and colorless complexes with Cu(I) in aqueous solution. Concluding from X-ray structural data, electrochemical measurements, and an extensive network of equilibrium titrations, all three ligands form discrete Cu(I) complexes with 1:1 stoichiometry and are capable of buffering Cu(I) concentrations between 10−10 and 10−17 M. As most Cu(I) protein affinities have been obtained from competition experiments with bathocuproine disulfonate (BCS) or 2,2′-bicinchoninic acid (BCA), we further calibrated their Cu(I) stability constants against the MCL-series. To demonstrate the application of these reagents, we determined the Cu(I) binding affinity of CusF (logK = 14.3±0.1), a periplasmic metalloprotein required for the detoxification of elevated copper levels in E. coli. Altogether, this interconnected set of affinity standards establishes a reliable foundation that will facilitate the precise determination of Cu(I) binding affinities of proteins and small molecule ligands. PMID:24298878